丙型肝炎病毒感染树鼩的实验研究
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摘要
长期以来,缺乏感染模型是丙型肝炎病毒(HCV)研究中所面临的难题,近两年来虽然建立了感染性的HCV细胞培养模型,但仅限于有限的基因型。由于缺乏理想的HCV感染小动物模型,HCV防治研究进展缓慢。HCV感染的标准疗法是聚乙二醇化的IFN-α与利巴韦林的联合应用,2、3型HCV感染对该疗法的持续反应率达75%以上,但流行最广的1型HCV感染的持续反应率不到50%。此外,该疗法的费用昂贵,还具有副作用。HCV分子克隆以来,HCV疫苗在发达国家一直是研究热点,但迄今为止,尚无任何有效的疫苗问世。探索更加有效的HCV治疗方法,研发有效的HCV疫苗非常迫切。
黑猩猩(Pan troglodytes)是目前唯一被肯定的HCV易感动物,但使用黑猩猩作为实验动物的费用很高,且动物来源极为有限。近年来有Trimera小鼠和uPA-SCID小鼠被用作HCV感染模型,但两者的制备方法复杂,且免疫缺陷的特性限制了其在HCV免疫学研究方面的应用。
树鼩(tree shrews,tupaia belangeri)是一种与灵长目动物诸多特征相似的小动物,被用于人类多种疾病,包括HBV、HSV等病毒感染的实验动物模型研究。1998年首次有报道,用HCV RNA阳性的血清感染中国云南野生树鼩,部分可发生短期病毒血症和HCV抗体阳转,2007年再次有类似报道。尽管如此,目前对树鼩是否可用作HCV感染的动物模型还存在争议。
病毒体与宿主细胞表面受体的结合是病毒进入细胞的第一步,病毒与细胞表面相应受体的结合是病毒感染复制的始动环节。虽然HCV进入靶细胞的确切机制现在仍不十分清楚,但一系列确凿的证据表明这与HCV包膜糖蛋白E2与靶细胞表面的多种受体相关分子的相互作用有关,这些分子包括CD81、SR-BI、claudin-1(CLDN1)以及最近鉴定出的occludin(OCLN)。
从1997年到2008年,有一些研究表明HCV病人血浆能感染PTH(primary tupaia hepatocytes),能感染树鼩,但树鼩能否作为小动物模型但是仍然存在争议。使用HCV病人血浆作为病毒来源,则无法保证研究的连贯性和重复性。因此,除临床病毒株(病人血浆)外,使用来源稳定、背景一致、感染性高的细胞培养HCV病毒代替丙型肝炎病人血浆进行HCV动物模型的研究是最好的解决方法。
由于病毒特异性受体的表达和分布是影响病毒感染的宿主和组织亲嗜性的重要限制性因素,因此关于树鼩CD81、CLDN1、SR-BI以及OCLN功能的研究有着重要的意义。本研究克隆了树鼩的CD81、SR-BI、CLDN1以及OCLN基因,用一系列功能实验分析这些分子是否可介导HCV感染,观察了HCV假病毒(HCV pseudopatticles, HCVpp)和细胞培养产生的HCV(cell culture produced HCV, HCVcc)对PTH的感染性,并用HCVcc体内感染树鼩,检测树鼩血清中是否可产生HCV病毒血症。
方法:1.抽提PTH的总RNA,逆转录为cDNA,设计引物,扩增出树鼩CD81、SR-BI、CLDN1以及OCLN基因序列。2.构建原核表达质粒pET32a-tCD81 LEL(large extracellular loop),在大肠杆菌BL21中表达树鼩CD81 LEL的重组蛋白,用ELISA分析HCV包膜E2蛋白与树鼩CD81 LEL的结合活性。3.构建真核表达质粒pCI-tCD81、pCI-tSR-BI转染CHO细胞,用流式细胞术检测HCV E2蛋白与其结合活性。4.将树鼩CD81基因转染HepG2细胞,再用荧光素酶作为报告基因的假HCV颗粒(HCVpp-Luc)感染,检测荧光素酶在细胞中的表达。5.构建真核表达质粒pCI-tCLDN1,转染293T细胞,再用HCVpp-Luc感染,检测荧光素酶活性。6.将人CD81、SR-BI、CLDN1以及树鼩OCLN共转染CHO细胞,然后用HCVpp-Luc感染,检测报告基因的表达。7.分离培养PTH,用HCVpp-Luc感染,检测荧光素酶活性。8.细胞培养的HCVcc(J6/JFH1)感染PTH,western blot检测HCV蛋白的表达。9.用HCVcc(J6/JFH1)感染PTH连续培养3周,取不同时间的培养上清感染Huh7.5细胞,免疫荧光检测细胞内的HCV蛋白。10.HCVcc(J6/JFH1)感染树鼩(6只实验组,6只对照组),连续十次(1次/周)取血,realtime-PCR检测血清中HCV拷贝数,同时检测ALT水平的变化。
结果:1.扩增出树鼩CD81(GenBank: EF581831),CLDN1(GenBank: EF584564),SR-BI(GenBank:EF584564),OCLN(GenBank:FJ809937)编码基因。2.扩增出树鼩和人的CD81 LEL,并构建了原核表达质粒pET32a-tCD81 LEL、pET32a-hCD81 LEL,获得重组蛋白。ELISA法检测发现人与树鼩的CD81 LEL都能结合可溶性HCV包膜E2蛋白,且二者与HCV E2的结合活性相似。3.构建树鼩CD81真核表达质粒,转染CHO细胞,检测发现CD81分子可在CHO细胞表面表达,且CHO细胞表面的CD81分子能与可溶性HCV E2蛋白结合。4.人或树鼩的CD81表达质粒转染的HepG2细胞均可被HCVpp感染。5.构建人与树鼩的SR-BI的真核表达质粒,转染CHO细胞,流式细胞术检测显示HCV包膜E2蛋白能与表达在CHO细胞表面的SR-BI分子结合。6.转染人或树鼩CLDN1表达质粒到293T细胞,转染后的细胞能被HCVpp感染。7.共转染人CD81、SR-BI、CLDN1及树鼩OCLN后CHO细胞能被HCVpp感染。8.HCVpp和HCVcc可感染PTH,且HCVcc感染PTH可产生生产性感染。9.J6/JFH1 HCVcc感染的6只树鼩中,有2只血清中能间歇性检测到HCV RNA。
结论:1.树鼩CD81、SR-BI、CLDN1以及OCLN可介导HCV感染。2.HCVpp、HCVcc能感染PTH,并且HCVcc感染PTH能完成完整的病毒复制过程,产生感染性HCV颗粒。3.J6/JFH1 HCVcc可感染树鼩,产生病毒血症。
以上结果为用树鼩作为HCV小动物感染模型的可行性提供了依据。
Detailed analyses of HCV have been hampered by the lacks of stable small animal model for a long time. In the recent two years, although the infectious HCV cell model has been established, it is restricted in only one genotype, which brought difficulties to HCV preventing and curing research. The current therapy for HCV infection is PegIFN-αin combination with ribavirin. Although the sustained viral response rate for HCV genotype 2 and 3 is more than 75%, it is no more than 50% for HCV genotype 1, the most widespread type in the world. This treatment is expensive and has side effects. Since HCV was cloned, some European and American developed countries have been always trying to develop its vaccine, but so far yet, there is no effective vaccine available. Thus, the development of new therapies and effective vaccine for HCV infection is of great clinical and economic significance.
The chimpanzee (Pan troglodytes) is the only nonhuman host serving as a model for HCV infection. While HCV infection can be successfully studied in chimpanzees, these animal experiments are expensive and raise ethical issues. An alternative model for HCV infection is the tree shrew (tupaia belangeri), a small, squirrel-like mammal closely related to primates. Tree shrew has been shown to be susceptible to avariety of human viruses including herpes simplex, hepatitis B, and rotavirus. Two studies have demonstrated that T. belangeri can be infected in vivo with HCV. Some researches had shown that primary tupaia hepatocytes (PTH) can be successfully infected with serum or plasma derived from chronically HCV-infected humans. Incubation of PTH with native HCV from chronically HCV infected patients resulted in the production of infectious virus in vitro, indicating that PTH provide a model for the study of HCV infection and the functional assessment of HCV receptor candidates. Three interesting models for HCV infection: the immunotolerized rat model, Trimera mouse model and the uPA/SCID model, are being developed. Trimera mouse model involves the development of a chimeric mouse with a different source of tissue. Indeed, these three rodent models are really promising, although relatively complicated to use, but they present the un-questionable advantage of being much less expensive and easier to maintain and breed than primates. The first step of cell entry for virus is to attach to the receptors on the target cell surface. In this step the virus envelope protein plays an important role.Viruses initiate infection by attaching to molecules or receptors on the cell surface. Thus, major research efforts are focused on HCV receptors.
The exact mechanism of HCV cell entry is still not very clear. A series of conclusive evidences show that the HCV envelope glycoprotein 2 could bind to a variety of target cell surface receptors, among which CD81 is most improtant. CD81, also known as TAPA-1, is a member of the tetraspanin family, containing four transmembrane domains. It is a 26kD nonglycosylated cell surface protein and absolutely necessary for the HCV cell entry.
Strong evidence exists for the involvement of the HCV E2-binding proteins-CD81 and SR-BI in HCV entry. However, these molecules are insufficient for productive viral entry because some cell lines express CD81 and SR-BI, but do not support HCV entry. Most recently, CLDN1 was found to be required in HCV entry and confer susceptibility to HCV when ectopically expressed in non-hepatic cells. CLDN1 is one of the major proteins forming backbone of tight junctions. It has four transmembrane helices, two extracellular loops (EL).The EL1 of CLDN1 is critical for HCV entry and antibodies directed against an epitope inserted in the CLDN1 EL1 block HCV infection.
Since 1997, several publications reported that primary tupaia hepatocytes (PTH) or tree shrews can be successfully infected with serum or plasma derived from chronically HCV-infected humans. But it is still controversial that PTH provide a model and tree shrew can be used as small animal model for the study of HCV infection and the functional assessment of HCV receptor candidates.For most viruses, the receptor distribution in an individual is associated with tissue tropism. Many virus-receptor interactions determine the host range and therefore constitute an interspecies barrier.The interaction of a virus with its cellular receptor initiates a chain of dynamic events that will enable entry of the virus into the cell. HCV Infection of the host cell is initiated through interactions between the HCV gps and several cell surface molecules. Currently, four host molecules, CD81, SR-BI, CLDN1 and OCLN, are thought to be specific and required (co-)receptors for HCV entry.
In present study, we cloned the tree shrew CD81, SR-BI CLDN1 and OCLN. And we analyzed the functional role of the receptors for HCV binding and infection of PTH. using HCVpp and HCVcc models. We demonstrate that they represent important cell surface molecules mediating binding of the HCV E2 and infections to hepatocytes. Our research provide further possibility of that HCV infect tree shrew, in vivo and taking it as potential small animal model for the study of HCV infection.
HCVcc were used to inoculate adult tree shrew in vivo. Alanine aminotransferase (ALT) levels, HCV RNA and viral load were determined in the animals before and after inoculation (from week 1 to week 10, once a week).
Methods 1.RNA was extracted from liver tissue of adult tree shrews (T. belangeri). And RT-PCR was performed to amplify tree shrews CD81、SR-BI and CLDN1. 2 . TRX-hCD81 LEL and TRX-tCD81 LEL fusion proteins were expressed in Escherichia coli DH5a. And the interactions between them to HCV E2 were analyzed by ELISA. 3.CHO cells were transduced with human and tree shrew CD81. The interaction between CHO and HCV sE2 was analyzed by FCM. 4.HepG2 cells were transduced with human and tree shrew CD81. The infection was analyzed by HCVpp-Luc. 5.CHO cells were transduced with human and tree shrew SR-BI. The interaction between CHO and HCV E2 was analyzed by FCM. 6.HEK 293T cells were transduced with human and tree shrew CLDN1. The infection was analyzed by HCVpp-Luc and HCVpp-EGFP . 8.CHO cells were transduced with human CD81,SR-BI, CLDN1 and tree shrew OCLN. The infection was analyzed by HCVpp-Luc 7.Primary tupaia hepatocytes were separated and cultivate. The infection was analyzed by HCVpp-Luc. 9. The infectivity of supernatant in PTH was analyzed by immunofluorescence and Western blotting. 10. The tree shrews were inoculated with HCVcc(J6/JFH1). Alanine aminotransferase (ALT) levels, HCV RNA and viral load were analyzed in the animals before and after inoculation.
Results 1.We cloned tree shrew CD81(GenBank: EF581831),SR-BI(GenBank: EF584564),CLDN1(GenBank:EF584564)and OCLN(GenBank:FJ809937)ORF. The comparitive analysis of CD81,SR-BI, CLDN1 and OCLN deduced amino acid sequences between human and tree shrew showed the high degree of homology.
2.We cloned tree shrew CD81 LEL ORF and cloned them into pET32a vector. The recombinant protein was express in E.coli1 BL21. The comparitive analysis of CD81LEL binding activity to HCV sE2 between human and tree shrew by ELISA demonstrated that bacterially expressed tCD81 LEL fused to TRX is able to bind HCV E2 proteins as well as hCD81 LEL.
3.CHO cells transduced with human and tree shrew CD81 can interact with HCV sE2.
4.HCVpp can infect HepG2 cells transduced with human and tree shrew CD81.
5.CHO cells transduced with human and tree shrew SR-BI can interact with HCV sE2.
6.HCVpp can infect the 293T cells transfected tree shrew CLDN1 .
7.HCVpp can infect PTH.
8.CHO cells transduced with human CD81,SR-BI,CLDN1 and tree shrew OCLN can be infected by HCVpp.
9.HCVcc can infect PTH. And HCVcc-infected PTH generated infectious virus in supernatants culture.
10.HCV RNA can be detected form 2 in 6 tree shrews inoculated with HCVcc by realtime-PCR .
Conclutions:
1. Tree shrew CD81 LEL and SR-BI can bind to HCV E2. Tree shrew CD81, SR-BI, CLDN1 and OCLN can mediate HCV infection.
2. HCVpp and HCVcc can infect PTH .
3. In vivo infection of tree shrew with HCVcc is possible and tree shrew may be used as small animal model to research HCV infection.
黑猩猩(Pan troglodytes)是目前唯一被肯定的HCV易感动物,但使用黑猩猩作为实验动物的费用很高,且动物来源极为有限。近年来有Trimera小鼠和uPA-SCID小鼠被用作HCV感染模型,但两者的制备方法复杂,且免疫缺陷的特性限制了其在HCV免疫学研究方面的应用。
树鼩(tree shrews,tupaia belangeri)是一种与灵长目动物诸多特征相似的小动物,被用于人类多种疾病,包括HBV、HSV等病毒感染的实验动物模型研究。1998年首次有报道,用HCV RNA阳性的血清感染中国云南野生树鼩,部分可发生短期病毒血症和HCV抗体阳转,2007年再次有类似报道。尽管如此,目前对树鼩是否可用作HCV感染的动物模型还存在争议。
病毒体与宿主细胞表面受体的结合是病毒进入细胞的第一步,病毒与细胞表面相应受体的结合是病毒感染复制的始动环节。虽然HCV进入靶细胞的确切机制现在仍不十分清楚,但一系列确凿的证据表明这与HCV包膜糖蛋白E2与靶细胞表面的多种受体相关分子的相互作用有关,这些分子包括CD81、SR-BI、claudin-1(CLDN1)以及最近鉴定出的occludin(OCLN)。
从1997年到2008年,有一些研究表明HCV病人血浆能感染PTH(primary tupaia hepatocytes),能感染树鼩,但树鼩能否作为小动物模型但是仍然存在争议。使用HCV病人血浆作为病毒来源,则无法保证研究的连贯性和重复性。因此,除临床病毒株(病人血浆)外,使用来源稳定、背景一致、感染性高的细胞培养HCV病毒代替丙型肝炎病人血浆进行HCV动物模型的研究是最好的解决方法。
由于病毒特异性受体的表达和分布是影响病毒感染的宿主和组织亲嗜性的重要限制性因素,因此关于树鼩CD81、CLDN1、SR-BI以及OCLN功能的研究有着重要的意义。本研究克隆了树鼩的CD81、SR-BI、CLDN1以及OCLN基因,用一系列功能实验分析这些分子是否可介导HCV感染,观察了HCV假病毒(HCV pseudopatticles, HCVpp)和细胞培养产生的HCV(cell culture produced HCV, HCVcc)对PTH的感染性,并用HCVcc体内感染树鼩,检测树鼩血清中是否可产生HCV病毒血症。
方法:1.抽提PTH的总RNA,逆转录为cDNA,设计引物,扩增出树鼩CD81、SR-BI、CLDN1以及OCLN基因序列。2.构建原核表达质粒pET32a-tCD81 LEL(large extracellular loop),在大肠杆菌BL21中表达树鼩CD81 LEL的重组蛋白,用ELISA分析HCV包膜E2蛋白与树鼩CD81 LEL的结合活性。3.构建真核表达质粒pCI-tCD81、pCI-tSR-BI转染CHO细胞,用流式细胞术检测HCV E2蛋白与其结合活性。4.将树鼩CD81基因转染HepG2细胞,再用荧光素酶作为报告基因的假HCV颗粒(HCVpp-Luc)感染,检测荧光素酶在细胞中的表达。5.构建真核表达质粒pCI-tCLDN1,转染293T细胞,再用HCVpp-Luc感染,检测荧光素酶活性。6.将人CD81、SR-BI、CLDN1以及树鼩OCLN共转染CHO细胞,然后用HCVpp-Luc感染,检测报告基因的表达。7.分离培养PTH,用HCVpp-Luc感染,检测荧光素酶活性。8.细胞培养的HCVcc(J6/JFH1)感染PTH,western blot检测HCV蛋白的表达。9.用HCVcc(J6/JFH1)感染PTH连续培养3周,取不同时间的培养上清感染Huh7.5细胞,免疫荧光检测细胞内的HCV蛋白。10.HCVcc(J6/JFH1)感染树鼩(6只实验组,6只对照组),连续十次(1次/周)取血,realtime-PCR检测血清中HCV拷贝数,同时检测ALT水平的变化。
结果:1.扩增出树鼩CD81(GenBank: EF581831),CLDN1(GenBank: EF584564),SR-BI(GenBank:EF584564),OCLN(GenBank:FJ809937)编码基因。2.扩增出树鼩和人的CD81 LEL,并构建了原核表达质粒pET32a-tCD81 LEL、pET32a-hCD81 LEL,获得重组蛋白。ELISA法检测发现人与树鼩的CD81 LEL都能结合可溶性HCV包膜E2蛋白,且二者与HCV E2的结合活性相似。3.构建树鼩CD81真核表达质粒,转染CHO细胞,检测发现CD81分子可在CHO细胞表面表达,且CHO细胞表面的CD81分子能与可溶性HCV E2蛋白结合。4.人或树鼩的CD81表达质粒转染的HepG2细胞均可被HCVpp感染。5.构建人与树鼩的SR-BI的真核表达质粒,转染CHO细胞,流式细胞术检测显示HCV包膜E2蛋白能与表达在CHO细胞表面的SR-BI分子结合。6.转染人或树鼩CLDN1表达质粒到293T细胞,转染后的细胞能被HCVpp感染。7.共转染人CD81、SR-BI、CLDN1及树鼩OCLN后CHO细胞能被HCVpp感染。8.HCVpp和HCVcc可感染PTH,且HCVcc感染PTH可产生生产性感染。9.J6/JFH1 HCVcc感染的6只树鼩中,有2只血清中能间歇性检测到HCV RNA。
结论:1.树鼩CD81、SR-BI、CLDN1以及OCLN可介导HCV感染。2.HCVpp、HCVcc能感染PTH,并且HCVcc感染PTH能完成完整的病毒复制过程,产生感染性HCV颗粒。3.J6/JFH1 HCVcc可感染树鼩,产生病毒血症。
以上结果为用树鼩作为HCV小动物感染模型的可行性提供了依据。
Detailed analyses of HCV have been hampered by the lacks of stable small animal model for a long time. In the recent two years, although the infectious HCV cell model has been established, it is restricted in only one genotype, which brought difficulties to HCV preventing and curing research. The current therapy for HCV infection is PegIFN-αin combination with ribavirin. Although the sustained viral response rate for HCV genotype 2 and 3 is more than 75%, it is no more than 50% for HCV genotype 1, the most widespread type in the world. This treatment is expensive and has side effects. Since HCV was cloned, some European and American developed countries have been always trying to develop its vaccine, but so far yet, there is no effective vaccine available. Thus, the development of new therapies and effective vaccine for HCV infection is of great clinical and economic significance.
The chimpanzee (Pan troglodytes) is the only nonhuman host serving as a model for HCV infection. While HCV infection can be successfully studied in chimpanzees, these animal experiments are expensive and raise ethical issues. An alternative model for HCV infection is the tree shrew (tupaia belangeri), a small, squirrel-like mammal closely related to primates. Tree shrew has been shown to be susceptible to avariety of human viruses including herpes simplex, hepatitis B, and rotavirus. Two studies have demonstrated that T. belangeri can be infected in vivo with HCV. Some researches had shown that primary tupaia hepatocytes (PTH) can be successfully infected with serum or plasma derived from chronically HCV-infected humans. Incubation of PTH with native HCV from chronically HCV infected patients resulted in the production of infectious virus in vitro, indicating that PTH provide a model for the study of HCV infection and the functional assessment of HCV receptor candidates. Three interesting models for HCV infection: the immunotolerized rat model, Trimera mouse model and the uPA/SCID model, are being developed. Trimera mouse model involves the development of a chimeric mouse with a different source of tissue. Indeed, these three rodent models are really promising, although relatively complicated to use, but they present the un-questionable advantage of being much less expensive and easier to maintain and breed than primates. The first step of cell entry for virus is to attach to the receptors on the target cell surface. In this step the virus envelope protein plays an important role.Viruses initiate infection by attaching to molecules or receptors on the cell surface. Thus, major research efforts are focused on HCV receptors.
The exact mechanism of HCV cell entry is still not very clear. A series of conclusive evidences show that the HCV envelope glycoprotein 2 could bind to a variety of target cell surface receptors, among which CD81 is most improtant. CD81, also known as TAPA-1, is a member of the tetraspanin family, containing four transmembrane domains. It is a 26kD nonglycosylated cell surface protein and absolutely necessary for the HCV cell entry.
Strong evidence exists for the involvement of the HCV E2-binding proteins-CD81 and SR-BI in HCV entry. However, these molecules are insufficient for productive viral entry because some cell lines express CD81 and SR-BI, but do not support HCV entry. Most recently, CLDN1 was found to be required in HCV entry and confer susceptibility to HCV when ectopically expressed in non-hepatic cells. CLDN1 is one of the major proteins forming backbone of tight junctions. It has four transmembrane helices, two extracellular loops (EL).The EL1 of CLDN1 is critical for HCV entry and antibodies directed against an epitope inserted in the CLDN1 EL1 block HCV infection.
Since 1997, several publications reported that primary tupaia hepatocytes (PTH) or tree shrews can be successfully infected with serum or plasma derived from chronically HCV-infected humans. But it is still controversial that PTH provide a model and tree shrew can be used as small animal model for the study of HCV infection and the functional assessment of HCV receptor candidates.For most viruses, the receptor distribution in an individual is associated with tissue tropism. Many virus-receptor interactions determine the host range and therefore constitute an interspecies barrier.The interaction of a virus with its cellular receptor initiates a chain of dynamic events that will enable entry of the virus into the cell. HCV Infection of the host cell is initiated through interactions between the HCV gps and several cell surface molecules. Currently, four host molecules, CD81, SR-BI, CLDN1 and OCLN, are thought to be specific and required (co-)receptors for HCV entry.
In present study, we cloned the tree shrew CD81, SR-BI CLDN1 and OCLN. And we analyzed the functional role of the receptors for HCV binding and infection of PTH. using HCVpp and HCVcc models. We demonstrate that they represent important cell surface molecules mediating binding of the HCV E2 and infections to hepatocytes. Our research provide further possibility of that HCV infect tree shrew, in vivo and taking it as potential small animal model for the study of HCV infection.
HCVcc were used to inoculate adult tree shrew in vivo. Alanine aminotransferase (ALT) levels, HCV RNA and viral load were determined in the animals before and after inoculation (from week 1 to week 10, once a week).
Methods 1.RNA was extracted from liver tissue of adult tree shrews (T. belangeri). And RT-PCR was performed to amplify tree shrews CD81、SR-BI and CLDN1. 2 . TRX-hCD81 LEL and TRX-tCD81 LEL fusion proteins were expressed in Escherichia coli DH5a. And the interactions between them to HCV E2 were analyzed by ELISA. 3.CHO cells were transduced with human and tree shrew CD81. The interaction between CHO and HCV sE2 was analyzed by FCM. 4.HepG2 cells were transduced with human and tree shrew CD81. The infection was analyzed by HCVpp-Luc. 5.CHO cells were transduced with human and tree shrew SR-BI. The interaction between CHO and HCV E2 was analyzed by FCM. 6.HEK 293T cells were transduced with human and tree shrew CLDN1. The infection was analyzed by HCVpp-Luc and HCVpp-EGFP . 8.CHO cells were transduced with human CD81,SR-BI, CLDN1 and tree shrew OCLN. The infection was analyzed by HCVpp-Luc 7.Primary tupaia hepatocytes were separated and cultivate. The infection was analyzed by HCVpp-Luc. 9. The infectivity of supernatant in PTH was analyzed by immunofluorescence and Western blotting. 10. The tree shrews were inoculated with HCVcc(J6/JFH1). Alanine aminotransferase (ALT) levels, HCV RNA and viral load were analyzed in the animals before and after inoculation.
Results 1.We cloned tree shrew CD81(GenBank: EF581831),SR-BI(GenBank: EF584564),CLDN1(GenBank:EF584564)and OCLN(GenBank:FJ809937)ORF. The comparitive analysis of CD81,SR-BI, CLDN1 and OCLN deduced amino acid sequences between human and tree shrew showed the high degree of homology.
2.We cloned tree shrew CD81 LEL ORF and cloned them into pET32a vector. The recombinant protein was express in E.coli1 BL21. The comparitive analysis of CD81LEL binding activity to HCV sE2 between human and tree shrew by ELISA demonstrated that bacterially expressed tCD81 LEL fused to TRX is able to bind HCV E2 proteins as well as hCD81 LEL.
3.CHO cells transduced with human and tree shrew CD81 can interact with HCV sE2.
4.HCVpp can infect HepG2 cells transduced with human and tree shrew CD81.
5.CHO cells transduced with human and tree shrew SR-BI can interact with HCV sE2.
6.HCVpp can infect the 293T cells transfected tree shrew CLDN1 .
7.HCVpp can infect PTH.
8.CHO cells transduced with human CD81,SR-BI,CLDN1 and tree shrew OCLN can be infected by HCVpp.
9.HCVcc can infect PTH. And HCVcc-infected PTH generated infectious virus in supernatants culture.
10.HCV RNA can be detected form 2 in 6 tree shrews inoculated with HCVcc by realtime-PCR .
Conclutions:
1. Tree shrew CD81 LEL and SR-BI can bind to HCV E2. Tree shrew CD81, SR-BI, CLDN1 and OCLN can mediate HCV infection.
2. HCVpp and HCVcc can infect PTH .
3. In vivo infection of tree shrew with HCVcc is possible and tree shrew may be used as small animal model to research HCV infection.
引文
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