戊型肝炎分子流行病学及其病原与宿主细胞互作机制研究
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摘要
戊型肝炎(HE)是由戊型肝炎病毒(HEV)引起的经肠道传播的急性病毒性肝炎。中国已被列为因HE发病和死亡所致的经济负担最严重的国家之一。越来越多的报道显示HE是一种人兽共患病,可以跨种间感染与传播。现已经证实对HEV易感的动物包括猪、非人灵长类、野鹿、牛、山羊、马等。猪等家养动物与人类接触密切,携带有HEV的这些动物很有可能通过密切接触而传染给人类,给公共卫生构成很大威胁。开展HE的病原学、流行病学、致病机理等方面的研究具有重要意义。
HE的研究已经有了很大的进展,但还存在很多问题。(1)我国HEV分子流行病学虽然已经开展了许多研究,但是很多地区的相关研究尚属空白。而且由于HEV的流行还受地理环境及当地人民生活习惯等因素的影响,不同地区HEV的流行特征不尽相同。因此我国部分地区的HEV分子流行病学调查还有待开展。(2)HEV的天然宿主较多,除了家养动物猪之外,野生动物的HEV携带情况也值得重视,然而我国对HEV的野生动物宿主研究较少。(3) HEV跨种间传播分子机制仍不清楚,因此亟待找到人或动物细胞表面HEV的受体分子,为防控HEV跨种间感染与传播提供理论借鉴。(4)HEV在宿主细胞内复制的分子机制研究仍然是空白。
有鉴于此,本研究主要从HE的分子流行病学、HEV与宿主细胞的互作机制等方面开展研究,结果如下:
一:HE的分子流行病学研究
(1)野生动物中HEV携带情况调查
从上海及安徽地区的2个野生动物园采集了191份野生动物粪便样品。通过RT-nPCR方法检测样品中的HEV RNA。结果表明,上海地区的野生动物未检测到HEV的存在,而在安徽某野生动物园的38份样品中,有11份为HEV RNA阳性。序列分析表明该11个毒株均为基因4型,序列之间的同源性为96-100%,推测在该野生动物园的不同种野生动物之间发生了HEV的跨种间传播。尽管从该野生动物园采集的7份工作人员血清终未检测到HEV RNA的存在,但HEV抗体的ELISA检测表明,其中有5人为HEV抗体IgG或IgM阳性。说明该野生动物园中的人也可能受到了HEV的感染。
(2)皖北地区人群及猪群HEV分子流行病学研究
从皖北苏鲁豫皖交界地区的阜阳市、淮北市及宿州市采集了1476份人普通人血清,正常猪群554份粪便样品。血清样品用ELISA检测HEV抗体IgG及IgM, HEV抗体阳性的血清和猪粪便样品通过RT-nPCR方法检测HEV RNA。为了检验人血清中HEV抗体与猪群HEV毒株的反应性,通过Dot-Blot方法验证了20份IgG阳性及9份IgM阳性的人血清样品与2株猪HEV代表毒株的反应。结果显示,皖北地区普通人群HEV抗体的总阳性率为7.93%(117/1476);HEV在血清中的阳性率为0.95%(14/1476);正常猪群中HEV病毒携带率为7.0%(39/554)。14个人HEV毒株及39个猪HEV毒株经过测序后进行基于核苷酸序列的系统进化分析,结果显示该53个毒株均属于基因4型HEV,聚类为3个群;39个猪源HEV分成2个群,而14个人HEV毒株单独聚为1个群。由此推测该地区猪源HEV毒株不是人感染HE的感染源。本研究还克隆了该地区2株猪源及1株人源HEV代表毒株的病毒外壳蛋白全基因序列。序列分析显示,该3株HEV外壳蛋白基因全长均为2025bp,编码674个aa。基于病毒外壳蛋白基因的系统进化分析显示3个毒株并聚类在3个不同的亚群之中,说明有3个不同的HEV毒株同时在皖北地区流行。
(3)华东地区几株HEV毒株遗传特征分析
本研究对儿童粪便样品中分离的1株HEV毒株、上海某马场分离的1株基因3型HEV毒株、上海市猪群流行的HEV代表毒株进行了序列分析。另外结合本实验室分离的10株代表毒株,检索了GenBank中57株其他国家或地区代表毒株,进行系统进化分析以阐明华东地区HEV毒株与它们之间的遗传进化关系。结果表明,(1)本研究从236份儿童粪便样品中检测到1株HEV,克隆了该毒株外壳蛋白基因全长,系统金华分析表明该毒株为基因4型,与1株日本毒株AB197673亲缘关系最近,同源性为97%。(2)中国4个不同省/市发现基因3型HEV在猪群中流行。2007年中国上海地区首次发现基因3型HEV以来,本研究对来自河南、安徽、上海的猪群进行HEV基因3检测,发现基因3型不仅在上海猪群中流行,还在安徽及河南的猪群流行;同时GenBank中有研究者提交基因3型HEV序列,本研究对这些序列分析发现中国大陆地区的基因3型HEV与日本基因3型HEV毒株亲缘关系最近。(3)对上海市猪群HEV代表毒株的全基因组序列分析发现,该毒株的基因编码方式与以前的基因4型HEV不一样,而与基因3型HEV编码方式相同,即外壳蛋白编码660个aa,而不是674个aa。该结果被Western Blot进一步证实。
二:HEV与宿主细胞的互作机制的初步探索
(1)猪肝细胞表面HEV候选受体蛋白的鉴定
本实验利用VOPBA技术鉴定猪胃、十二指肠、肺、肝脏、肠系膜淋巴结、肾脏及脾脏组织细胞表面HEV候选受体。结果表明在肝组织及胃组织表面有一条约55kDa的目的条带,该蛋白条带纯化后可以显著阻断HEV对易感细胞A549的产生的细胞病变。质谱鉴定结果表明该蛋白条带成分复杂,推测其中内在膜蛋白2B、膜突蛋白-B、ATP合成酶肝脏异构体及细胞色素P450有可能是HEV的候选受体。
(2)肝cDNA文库中与HEV pORF3互作蛋白的筛选
HEV ORF3编码一个磷酸化蛋白,但是在HEV感染宿主细胞中行使的具体的功能尚不明确,本研究利用CytoTrap酵母双杂交系统,以pORF3作为诱饵蛋白,在人肝cDNA表达文库中筛选与HEV pORF3相互作用蛋白。结果从肝cDNA表达文库中筛选到了10个与HEV pORF3相互作用蛋白,为进一步研究pORF3在HEV感染宿主细胞中的功能奠定了一定的基础。
(3)HEV感染A549细胞的比较蛋白质组学研究
本研究利用双向电泳结合质谱鉴定比较了HEV感染A549细胞与对照攻毒细胞的蛋白质组差异。HEV感染A549细胞后,经细胞病变观察、Dot-blot、RT-nPCR、免疫电镜验证HEV病毒感染细胞后,提取细胞总蛋白,双向电泳分离试验组细胞与对照组细胞总蛋白,分析蛋白点的差异。结果表明HEV感染试验组与对照组细胞总蛋白共有31个显著差异点,其中21个点在HEV感染A549细胞中表达丰度降低,10个点在HEV感染A549细胞中表达丰度升高。质谱鉴定表明31个点中的28个点有可靠的肽指纹图谱及可信搜库结果,这些蛋白包括代谢相关蛋白为9个,细胞骨架蛋白6个,细胞内大分子处理类蛋白6个,细胞信号转导成份2个,膜泡转运相关蛋白3个,细胞增殖相关蛋白1个,病毒外壳蛋白点1个。RT-nPCR方法检测其中27个宿主细胞基因在实验组细胞和对照攻毒细胞内mRNA水平的表达差异,结果表明15个基因有有效PCR扩增结果,其中8个与比较蛋白质组学结果一致。从27个蛋白点中选择1个表达上调和1个表达下调的蛋白用Western Blot方法进一步验证,显示与比较蛋白质组学结果一致。说明比较蛋白质学结果可信。由此推测,HEV感染细胞后导致细胞代谢减缓并抑制了细胞骨架的组装,为病毒在细胞内复制创造有利条件。
Hepatitis E (HE) is an important public health problem in developing countries. The hepatitis E virus (HEV), a member of the genus Hepevirus, is the causative agent of HE. HEV is a non-enveloped virus with a positives-stranded RNA genome approximately 7.2 kb in length and contains three open reading frames (ORFs). HEV isolates can be divided into four distinct genotypes according to the phylogenetic analysis based on nucleotide sequences.
HEV is believed to be transmitted by the faecal-oral route, and outbreaks of hepatitis E are attributed to water contaminated with HEV. HEV and antibodies to HEV have been reportedly found in a wide variety of animals, especially swine. Animals may act as an important role in the cross-species transmission of HEV. China owns large number of wild-life animal sources, but the infection status of HEV in this group is still unclear. Since 2000, genotype 4 HEV has become the dominant cause of hepatitis E disease in China. A recent report showed that genotype 4 HEV is freely transmitted between humans and swine in eastern China
Although the great progress have been made in the research field of HEV, there are still some unclear points, including (1) The interaction mechanism between HEV and its host cell; (2) The replication mechanism of HEV in host cell; (3) The translation of HEV in host cells;
(4) Pathogenesis of HEV.
In order to elucidate some points mentioned above, we performed the following research:
1. Study of the molecular epidemiology of HE.
(1) Detection of HEV in the wild-life animals 191 fecal samples were collected from two wild-life zoo lie in Shanghai city and Anhui Province, respectively. The HEV RNA was detected by using RT-nPCR method. The results indicated that 11 of 39 fecal samples collected from Anhui Province were positive for HEV RNA. Sequence analysis showed that all the 11 HEV strains belonged to genotype 4, sharing more than 96-100% sequence identities with each other, which suggested that cross-species infection of HEV happened in the wild-life zoo. Moreover, 5of 7 serum samples collected from the workers in the zoo were positive for anti-HEV antibodies, suggesting that human in the zoo were also infected by this HEV strain, though we had not isolate HEV RNA from these sera.
(2) Prevalence of HEV in the human and swine populations in the north part of Anhui Province
The prevalence of HEV is related with season, geography environment, and the human living habit. More and more cases of HEV cross-species infection have been reported all over the world. The north part of Anhui province is a farming area where swine farms rich distribute. We collected 1476 human sera from general human population and 554 fecal samples from general swine groups from this area. The serum samples were detected for the prevalence of anti-HEV antibodies and the antibody-positive sera were further detected for HEV RNA. The fecal samples were tested for the HEV RNA. Our results indicated that 7.93% (117/1476) of the sera were positive for anti-HEV IgG or IgM, 0.95% (14/1476) of the sera were positive for HEV RNA. The HEV positive rate of the fecal samples was 7.0% (39/554). Sequence analysis of the 53 strains showed that these strains clustered into 3 main groups, the strains from pigs were divided into 2 groups, and all the strains isolated from human population clustered together, forming a group without swine HEV strains, which suggested that no cross-human-swine infection of HEV were involved into this area. The full-length ORF2 gene of 3 representative HEV strains in the 3 main groups were determined and sequence analysis indicated all of them contain 2025 bp, encoding a putative 674 aa proteins. Phylogenetic analysis showed that these 3 strains clustered into 3 genetically distinct groups.
(3) Isolation and characterization of several HEV strains in eastern China
This study analyzed the HEV strains isolated from an infant, horse, shanghai swine and other 67 HEV strains we cloned or referenced from GenBank. The results suggested the following contents: (1) This is the first time that HEV strain was isolated from an infant less than one-year-old, the full-length ORF2 gene of this strain was determined and shared 97% sequence identity with a Japanese HEV strain, AB197673; (2) A genotype 3 HEV strain was detected in one horse serum, and this HEV strain clustered closely with a genotype 3 HEV strain isolated from a pig in Zhejiang province; (3) Analysis of the complete genome of Shanghai swine HEV strain indicated that the genome organization of it was identical to genotype 1-3, but inconsistent with previous genotype 4 HEV strains, that is, ORF2 of this shanghai swine HEV strain codes 660aa not 674 aa. This result was confirmed by western blot assay.
2. The mechanism of HEV interation with the host cell
(1) Identification of a 55kDa protein as a candidate receptor of HEV on liver cell of pig.
In the present study, we try to find the candidate receptor of HEV on cell surface of different tissues, including stomach, dodecadactylon, lung, liver, mesenteric lymph node, kidney, and spleen of pig, using virus overlap proteins binding assay (VOPBA). The results indicated that a 55kDa aimed protein band was identified on the liver and stamoch cell surface, and this protein band was purified and could significantly reduce the CPE of A549 caused by HEV infection. The protein band was subjected to Mass spectrum assay, and the results showed the components of this protein band were very complicated and radixin, integral membrane protein 2B, ATP synthase subunit alpha liver isoform, and cytochrome P450 2C49 may be the candidate receptor of HEV on the pig liver cell surface.
(2) Screening of proteins that interact with HEV pORF3 in the human liver cDNA expression library using CytoTrap yeast two-hybrid system.
So far, there has no good cell system for HEV culture, which greatly hinders the study of the mechanism of HEV interaction with its host cell. The true function of pORF3 of HEV is still unclear. In the present study, using CytoTrap yeast two-hybrid system, we screened 10 proteins which interacted with HEV pORF3, which will be good base for elucidating the function of HEV pORF3 in the process of HEV infection its host cells.
(3) Te comparative proteomic study of A549 cell infected with HEV.
The molecular mechanism of interaction between HEV and host cell is still unclear. In the present study, combining 2-dismension electrophoresis (2-DE) and MALDI-TOF, we compared the protein expression of the HEV infected A549 cell and control cell. After the A549 cells were incubated with HEV, cytopathic effect assay, dot-blot assay, RT-PCR and immune electronic microscope (IEM) were used to confirm that the A549 cells were infected by HEV. The total proteins of the experimental and control cell were extracted and subjected to 2-DE and the protein dots which showed different between experimental and control cell were sliced from the gel and were analyzed by MALDI-TOF. The results indicated that 31 protein dots showed difference between experimental and control cells. Nine of them were related to metabolism of big molecular in cell, 6 of them were components of cytoskeleton, 6 of them join in molecular modification in cell, 4 of them join in the process of membrane bubble transmission, 2 of them joined in the process of signal transduction, and one of them was the capsid protein of HEV. The mRNA expression of 15 of them was identified using RT-PCR method, the protein expression of two protein dots were confirmed by western blot assay. This suggested that the compared proteomic results were convinced. From the results of compared proteome, we speculated that HEV infection reduced the speed of metabolism and the assembly of cytoskeleton so as to provide suitable environment for HEV replication.
HE的研究已经有了很大的进展,但还存在很多问题。(1)我国HEV分子流行病学虽然已经开展了许多研究,但是很多地区的相关研究尚属空白。而且由于HEV的流行还受地理环境及当地人民生活习惯等因素的影响,不同地区HEV的流行特征不尽相同。因此我国部分地区的HEV分子流行病学调查还有待开展。(2)HEV的天然宿主较多,除了家养动物猪之外,野生动物的HEV携带情况也值得重视,然而我国对HEV的野生动物宿主研究较少。(3) HEV跨种间传播分子机制仍不清楚,因此亟待找到人或动物细胞表面HEV的受体分子,为防控HEV跨种间感染与传播提供理论借鉴。(4)HEV在宿主细胞内复制的分子机制研究仍然是空白。
有鉴于此,本研究主要从HE的分子流行病学、HEV与宿主细胞的互作机制等方面开展研究,结果如下:
一:HE的分子流行病学研究
(1)野生动物中HEV携带情况调查
从上海及安徽地区的2个野生动物园采集了191份野生动物粪便样品。通过RT-nPCR方法检测样品中的HEV RNA。结果表明,上海地区的野生动物未检测到HEV的存在,而在安徽某野生动物园的38份样品中,有11份为HEV RNA阳性。序列分析表明该11个毒株均为基因4型,序列之间的同源性为96-100%,推测在该野生动物园的不同种野生动物之间发生了HEV的跨种间传播。尽管从该野生动物园采集的7份工作人员血清终未检测到HEV RNA的存在,但HEV抗体的ELISA检测表明,其中有5人为HEV抗体IgG或IgM阳性。说明该野生动物园中的人也可能受到了HEV的感染。
(2)皖北地区人群及猪群HEV分子流行病学研究
从皖北苏鲁豫皖交界地区的阜阳市、淮北市及宿州市采集了1476份人普通人血清,正常猪群554份粪便样品。血清样品用ELISA检测HEV抗体IgG及IgM, HEV抗体阳性的血清和猪粪便样品通过RT-nPCR方法检测HEV RNA。为了检验人血清中HEV抗体与猪群HEV毒株的反应性,通过Dot-Blot方法验证了20份IgG阳性及9份IgM阳性的人血清样品与2株猪HEV代表毒株的反应。结果显示,皖北地区普通人群HEV抗体的总阳性率为7.93%(117/1476);HEV在血清中的阳性率为0.95%(14/1476);正常猪群中HEV病毒携带率为7.0%(39/554)。14个人HEV毒株及39个猪HEV毒株经过测序后进行基于核苷酸序列的系统进化分析,结果显示该53个毒株均属于基因4型HEV,聚类为3个群;39个猪源HEV分成2个群,而14个人HEV毒株单独聚为1个群。由此推测该地区猪源HEV毒株不是人感染HE的感染源。本研究还克隆了该地区2株猪源及1株人源HEV代表毒株的病毒外壳蛋白全基因序列。序列分析显示,该3株HEV外壳蛋白基因全长均为2025bp,编码674个aa。基于病毒外壳蛋白基因的系统进化分析显示3个毒株并聚类在3个不同的亚群之中,说明有3个不同的HEV毒株同时在皖北地区流行。
(3)华东地区几株HEV毒株遗传特征分析
本研究对儿童粪便样品中分离的1株HEV毒株、上海某马场分离的1株基因3型HEV毒株、上海市猪群流行的HEV代表毒株进行了序列分析。另外结合本实验室分离的10株代表毒株,检索了GenBank中57株其他国家或地区代表毒株,进行系统进化分析以阐明华东地区HEV毒株与它们之间的遗传进化关系。结果表明,(1)本研究从236份儿童粪便样品中检测到1株HEV,克隆了该毒株外壳蛋白基因全长,系统金华分析表明该毒株为基因4型,与1株日本毒株AB197673亲缘关系最近,同源性为97%。(2)中国4个不同省/市发现基因3型HEV在猪群中流行。2007年中国上海地区首次发现基因3型HEV以来,本研究对来自河南、安徽、上海的猪群进行HEV基因3检测,发现基因3型不仅在上海猪群中流行,还在安徽及河南的猪群流行;同时GenBank中有研究者提交基因3型HEV序列,本研究对这些序列分析发现中国大陆地区的基因3型HEV与日本基因3型HEV毒株亲缘关系最近。(3)对上海市猪群HEV代表毒株的全基因组序列分析发现,该毒株的基因编码方式与以前的基因4型HEV不一样,而与基因3型HEV编码方式相同,即外壳蛋白编码660个aa,而不是674个aa。该结果被Western Blot进一步证实。
二:HEV与宿主细胞的互作机制的初步探索
(1)猪肝细胞表面HEV候选受体蛋白的鉴定
本实验利用VOPBA技术鉴定猪胃、十二指肠、肺、肝脏、肠系膜淋巴结、肾脏及脾脏组织细胞表面HEV候选受体。结果表明在肝组织及胃组织表面有一条约55kDa的目的条带,该蛋白条带纯化后可以显著阻断HEV对易感细胞A549的产生的细胞病变。质谱鉴定结果表明该蛋白条带成分复杂,推测其中内在膜蛋白2B、膜突蛋白-B、ATP合成酶肝脏异构体及细胞色素P450有可能是HEV的候选受体。
(2)肝cDNA文库中与HEV pORF3互作蛋白的筛选
HEV ORF3编码一个磷酸化蛋白,但是在HEV感染宿主细胞中行使的具体的功能尚不明确,本研究利用CytoTrap酵母双杂交系统,以pORF3作为诱饵蛋白,在人肝cDNA表达文库中筛选与HEV pORF3相互作用蛋白。结果从肝cDNA表达文库中筛选到了10个与HEV pORF3相互作用蛋白,为进一步研究pORF3在HEV感染宿主细胞中的功能奠定了一定的基础。
(3)HEV感染A549细胞的比较蛋白质组学研究
本研究利用双向电泳结合质谱鉴定比较了HEV感染A549细胞与对照攻毒细胞的蛋白质组差异。HEV感染A549细胞后,经细胞病变观察、Dot-blot、RT-nPCR、免疫电镜验证HEV病毒感染细胞后,提取细胞总蛋白,双向电泳分离试验组细胞与对照组细胞总蛋白,分析蛋白点的差异。结果表明HEV感染试验组与对照组细胞总蛋白共有31个显著差异点,其中21个点在HEV感染A549细胞中表达丰度降低,10个点在HEV感染A549细胞中表达丰度升高。质谱鉴定表明31个点中的28个点有可靠的肽指纹图谱及可信搜库结果,这些蛋白包括代谢相关蛋白为9个,细胞骨架蛋白6个,细胞内大分子处理类蛋白6个,细胞信号转导成份2个,膜泡转运相关蛋白3个,细胞增殖相关蛋白1个,病毒外壳蛋白点1个。RT-nPCR方法检测其中27个宿主细胞基因在实验组细胞和对照攻毒细胞内mRNA水平的表达差异,结果表明15个基因有有效PCR扩增结果,其中8个与比较蛋白质组学结果一致。从27个蛋白点中选择1个表达上调和1个表达下调的蛋白用Western Blot方法进一步验证,显示与比较蛋白质组学结果一致。说明比较蛋白质学结果可信。由此推测,HEV感染细胞后导致细胞代谢减缓并抑制了细胞骨架的组装,为病毒在细胞内复制创造有利条件。
Hepatitis E (HE) is an important public health problem in developing countries. The hepatitis E virus (HEV), a member of the genus Hepevirus, is the causative agent of HE. HEV is a non-enveloped virus with a positives-stranded RNA genome approximately 7.2 kb in length and contains three open reading frames (ORFs). HEV isolates can be divided into four distinct genotypes according to the phylogenetic analysis based on nucleotide sequences.
HEV is believed to be transmitted by the faecal-oral route, and outbreaks of hepatitis E are attributed to water contaminated with HEV. HEV and antibodies to HEV have been reportedly found in a wide variety of animals, especially swine. Animals may act as an important role in the cross-species transmission of HEV. China owns large number of wild-life animal sources, but the infection status of HEV in this group is still unclear. Since 2000, genotype 4 HEV has become the dominant cause of hepatitis E disease in China. A recent report showed that genotype 4 HEV is freely transmitted between humans and swine in eastern China
Although the great progress have been made in the research field of HEV, there are still some unclear points, including (1) The interaction mechanism between HEV and its host cell; (2) The replication mechanism of HEV in host cell; (3) The translation of HEV in host cells;
(4) Pathogenesis of HEV.
In order to elucidate some points mentioned above, we performed the following research:
1. Study of the molecular epidemiology of HE.
(1) Detection of HEV in the wild-life animals 191 fecal samples were collected from two wild-life zoo lie in Shanghai city and Anhui Province, respectively. The HEV RNA was detected by using RT-nPCR method. The results indicated that 11 of 39 fecal samples collected from Anhui Province were positive for HEV RNA. Sequence analysis showed that all the 11 HEV strains belonged to genotype 4, sharing more than 96-100% sequence identities with each other, which suggested that cross-species infection of HEV happened in the wild-life zoo. Moreover, 5of 7 serum samples collected from the workers in the zoo were positive for anti-HEV antibodies, suggesting that human in the zoo were also infected by this HEV strain, though we had not isolate HEV RNA from these sera.
(2) Prevalence of HEV in the human and swine populations in the north part of Anhui Province
The prevalence of HEV is related with season, geography environment, and the human living habit. More and more cases of HEV cross-species infection have been reported all over the world. The north part of Anhui province is a farming area where swine farms rich distribute. We collected 1476 human sera from general human population and 554 fecal samples from general swine groups from this area. The serum samples were detected for the prevalence of anti-HEV antibodies and the antibody-positive sera were further detected for HEV RNA. The fecal samples were tested for the HEV RNA. Our results indicated that 7.93% (117/1476) of the sera were positive for anti-HEV IgG or IgM, 0.95% (14/1476) of the sera were positive for HEV RNA. The HEV positive rate of the fecal samples was 7.0% (39/554). Sequence analysis of the 53 strains showed that these strains clustered into 3 main groups, the strains from pigs were divided into 2 groups, and all the strains isolated from human population clustered together, forming a group without swine HEV strains, which suggested that no cross-human-swine infection of HEV were involved into this area. The full-length ORF2 gene of 3 representative HEV strains in the 3 main groups were determined and sequence analysis indicated all of them contain 2025 bp, encoding a putative 674 aa proteins. Phylogenetic analysis showed that these 3 strains clustered into 3 genetically distinct groups.
(3) Isolation and characterization of several HEV strains in eastern China
This study analyzed the HEV strains isolated from an infant, horse, shanghai swine and other 67 HEV strains we cloned or referenced from GenBank. The results suggested the following contents: (1) This is the first time that HEV strain was isolated from an infant less than one-year-old, the full-length ORF2 gene of this strain was determined and shared 97% sequence identity with a Japanese HEV strain, AB197673; (2) A genotype 3 HEV strain was detected in one horse serum, and this HEV strain clustered closely with a genotype 3 HEV strain isolated from a pig in Zhejiang province; (3) Analysis of the complete genome of Shanghai swine HEV strain indicated that the genome organization of it was identical to genotype 1-3, but inconsistent with previous genotype 4 HEV strains, that is, ORF2 of this shanghai swine HEV strain codes 660aa not 674 aa. This result was confirmed by western blot assay.
2. The mechanism of HEV interation with the host cell
(1) Identification of a 55kDa protein as a candidate receptor of HEV on liver cell of pig.
In the present study, we try to find the candidate receptor of HEV on cell surface of different tissues, including stomach, dodecadactylon, lung, liver, mesenteric lymph node, kidney, and spleen of pig, using virus overlap proteins binding assay (VOPBA). The results indicated that a 55kDa aimed protein band was identified on the liver and stamoch cell surface, and this protein band was purified and could significantly reduce the CPE of A549 caused by HEV infection. The protein band was subjected to Mass spectrum assay, and the results showed the components of this protein band were very complicated and radixin, integral membrane protein 2B, ATP synthase subunit alpha liver isoform, and cytochrome P450 2C49 may be the candidate receptor of HEV on the pig liver cell surface.
(2) Screening of proteins that interact with HEV pORF3 in the human liver cDNA expression library using CytoTrap yeast two-hybrid system.
So far, there has no good cell system for HEV culture, which greatly hinders the study of the mechanism of HEV interaction with its host cell. The true function of pORF3 of HEV is still unclear. In the present study, using CytoTrap yeast two-hybrid system, we screened 10 proteins which interacted with HEV pORF3, which will be good base for elucidating the function of HEV pORF3 in the process of HEV infection its host cells.
(3) Te comparative proteomic study of A549 cell infected with HEV.
The molecular mechanism of interaction between HEV and host cell is still unclear. In the present study, combining 2-dismension electrophoresis (2-DE) and MALDI-TOF, we compared the protein expression of the HEV infected A549 cell and control cell. After the A549 cells were incubated with HEV, cytopathic effect assay, dot-blot assay, RT-PCR and immune electronic microscope (IEM) were used to confirm that the A549 cells were infected by HEV. The total proteins of the experimental and control cell were extracted and subjected to 2-DE and the protein dots which showed different between experimental and control cell were sliced from the gel and were analyzed by MALDI-TOF. The results indicated that 31 protein dots showed difference between experimental and control cells. Nine of them were related to metabolism of big molecular in cell, 6 of them were components of cytoskeleton, 6 of them join in molecular modification in cell, 4 of them join in the process of membrane bubble transmission, 2 of them joined in the process of signal transduction, and one of them was the capsid protein of HEV. The mRNA expression of 15 of them was identified using RT-PCR method, the protein expression of two protein dots were confirmed by western blot assay. This suggested that the compared proteomic results were convinced. From the results of compared proteome, we speculated that HEV infection reduced the speed of metabolism and the assembly of cytoskeleton so as to provide suitable environment for HEV replication.
引文
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