【作者】 赵钦；

【导师】 周恩民；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2011， 博士

【摘要】 禽戊型肝炎病毒(Hepatitis E virus,HEV)是鸡的大肝大脾(Big liver and spleen disease,BLS)病或肝炎脾大(Hepatitis-Splenomegaly,HS)综合症的主要病原,主要引起30-72周龄的蛋鸡和肉种鸡的死淘率升高和产蛋率下降(20-40%),发病鸡通常腹部充血,卵巢退化,肝脏脂肪或淀粉样变性,偶有肝脾肿大。该病曾给澳大利亚的养禽业造成了严重的经济损失,而我国也时有该病爆发的报道。禽HEV与人、猪HEV同属于肝炎病毒属,为无囊膜,单股正链RNA病毒,其基因组全长约为6.6kb,比哺乳动物HEV基因组全长少600bp左右,包含5’帽子和3’PolyA结构,含有3个开放阅读框(Open reading frame,ORF),分别为ORF1、ORF2和ORF3。其中ORF2基因编码衣壳蛋白是病毒的主要结构蛋白,并且包含病毒主要的抗原表位。由于禽HEV没有合适有效的体外培养系统,因此对该病毒的研究大多主要集中在病毒的亚单位蛋白ORF2上,通过原核或真核表达该蛋白,了解该蛋白的生物学活性和抗原性等,从而为该病毒的诊断和预防等提供材料和理论支持。1.国内禽HEV的RT-PCR检测根据实验室前期的禽HEV的血清学调查结果,我们从山东部分的禽HEV抗体阳性率较高的鸡场,收集患有BLS病或HS综合症病鸡的粪便、胆汁和血清。然后参考国外研究学者设计的禽HEV检测引物,对收集样品中的禽HEV RNA进行RT-PCR检测。结果从山东某肉种鸡场收集的粪便和胆汁样品中检测到禽HEV ORF2部分序列,通过该段序列与国外参考序列的同源性比较发现,与欧洲部分国家的分离株同源性较高。2.国内禽HEV分离株的全基因组序列分析参考GenBank上已知禽HEV的全基因组序列,设计12对引物。利用RT-nest-PCR方法从禽HEV核酸检测阳性的胆汁中,扩增六段相互重叠的禽HEV的基因组片段,然后通过软件拼接获得首个国内禽HEV分离株的全基因组序列,命名为CaHEV。将CaHEV与已知参考的4个禽HEV全基因组序列进行同源性和进化树分析,结果发现CaHEV与欧洲同源性最高,同属于禽HEV基因3型。并且不同禽HEV基因型的ORF2之间,氨基酸同源性达到97%以上,提示禽HEV可能仅存在单一的血清型。3. CaHEV的致病性研究将含有禽HEV的胆汁翅静脉攻毒1周龄的SPF鸡,对病毒进行增殖,然后用GE方法对病毒悬液进行定量。利用定量的CaHEV病毒悬液,通过口腔和翅静脉两种途径分别攻毒15周龄的SPF鸡,每组26只。攻毒后每天收集粪样,每三天收集血清,每周剖杀两只鸡,直到第12周剖杀所有鸡只,收集剖杀鸡只的胆汁,肝脏和脾脏同时观察剖杀鸡只的肝脏和脾脏的肉眼损害以及其病理学变化。通过对收集样品的RT-PCR检测发现,翅静脉攻毒组,攻毒后第3天就能从粪便中检测到病毒,一直持续到攻毒后54天仍能检测到;第6天开始出现病毒血症,约持续到第21天左右;口腔攻毒组,在攻毒后第6天开始粪便排毒,第62天仍然排毒,病毒血症开始出现在第12天,持续到第33天。通过ELISA检测攻毒鸡的禽HEV抗体发现,翅静脉攻毒组,抗体效价迅速达到高峰后就开始消退,而口腔接种组缓慢达到高峰并维持一段时间。另外,两种途径接种均有只2只鸡出现了肝脾肿大和严重的出血坏死,脏器的病理学变化也都符合HS综合症的部分临床症状,实验进一步论证了禽HEV可能是HS综合症的主要病原,但并不是唯一原因。3.禽HEV抗体检测的间接ELISA方法的建立及应用利用大肠杆菌原核表达禽HEV衣壳蛋白C端268个氨基酸,纯化后获得蛋白ORF2-268,将该蛋白作为包被抗原,摸索优化ELISA条件,建立禽HEV血清学诊断的间接ELISA方法。利用该ELISA方法,调查了山东省11个种鸡场的禽HEV感染情况。结果发现11个种鸡场都有过禽HEV的感染,不同鸡场的血清阳性率差异显著。其中有两个鸡场的阳性率达到了77%左右,而在检测的所有1379份血清中,493份阳性,阳性率也达到了35.8%,说明禽HEV的感染在山东省某些种鸡场已经非常严重,而且该病的感染已在全省范围内广泛流行。此外,利用该ELISA方法,跟踪检测了某种鸡场5个不同厂区的不同周龄鸡的禽HEV抗体的阳性率,结果发现,5个厂区都在12周龄左右就检测到了禽HEV抗体,在20-30周龄时,抗体阳性率达到最大,其中某个厂区在40-50周龄时出现了第二个高峰,大于60周龄时,抗体阳性率非常低。结果说明禽HEV在该种鸡场自然状况下的感染通常发生在小于10周龄的鸡,当鸡只达到20-30周龄时,禽HEV感染了鸡场里大多数的鸡,当达到60周龄时,禽HEV抗体已经在大多数鸡中消退。4.禽HEV人工感染鸡后产生的针对不同抗原区的抗体变化规律禽HEV的衣壳蛋白含有病毒主要的抗原表位,并且通过软件分析和前期实验室的研究发现,衣壳蛋白的前半段22-83aa存在着一个主要的抗原区,而在后半段有339-383aa,389-410aa,461-492aa,556-566aa和583-600aa 5个主要的抗原区,另外ORF3蛋白上也存在着抗原表位能够刺激机体产生免疫反应。为了研究这几个抗原区在人工感染鸡中产生的免疫应答情况,我们利用实验室前期表达的不同的截段蛋白ORF2-F(包含22-83aa),ORF2-S(包含后半段5个主要的抗原区),ORF2-5(339-442aa,包含后半段前两个抗原区),ORF2-8(383-515aa,包含后半段第2和第3个抗原区),ORF2-9(412-546aa,包含第4个抗原区)和ORF3蛋白作为包被抗原,间接ELISA检测了禽HEV感染鸡后不同日龄的血清中针对不同抗原区的抗体变化规律。结果发现针对ORF2-S蛋白的抗体在感染后12天左右就可以检测到,比ORF2-F和ORF3的早约3天左右,但是针对ORF2-F和ORF3的抗体效价迅速达到高峰后(感染后21天左右)就开始消退,而针对ORF2-S的则缓慢达到高峰维持一段时间后,缓慢消退。用ORF2-5作为包被抗原检测的抗体效价变化规律与用ORF2-S的相似,而用ORF2-8作为包被抗原的则测得抗体效价很低,说明人工感染禽HEV的鸡主要产生了针对抗原区339-383aa的抗原应答反应。当用ORF2-9作为包被抗原时,检测不到任何针对该蛋白的抗体,说明人工感染禽HEV的鸡并没有对该区域412-546aa产生免疫应答反应。5.禽HEV病毒样粒子的获得利用杆状病毒真核表达系统,表达了禽HEV衣壳蛋白全长,N端缺失56个氨基酸的ORF2Δ56和C端268个氨基酸的ORF2-268蛋白。通过IFA和Western-blot鉴定发现,三段蛋白都在该系统中成功表达,并且发现ORF2Δ56蛋白表达后分泌到细胞培养上清中。同时将该系统表达的三段蛋白与原核表达的相同氨基酸序列的蛋白分析发现,该系统除了表达获得预期大小的蛋白外,还获得了多个比预期大小小的蛋白,证实了禽HEV衣壳蛋白在该系统中表达可能被细胞内的酶酶解成小的片段。另外,通过对分泌到细胞上清中的ORF2Δ56蛋白蔗糖密度梯度离心纯化,负染透射电镜观察,发现禽HEV衣壳蛋白的片段ORF2Δ56蛋白在sf9昆虫细胞中表达后可以自我组装成病毒样粒子。更多还原

【Abstract】 Avian hepatitis E virus (HEV), the causative agent of big liver and spleen (BLS) disease or hepatitis-splenomegaly (HS) syndrome, mainly infects 30-70 week old aged broiler breeder hens and laying hens and causes increasing mortality, egg reduction (20-40%), red fluid in the abdomen, regressive ovaries and enlarged liver and spleen in chickens. The diseases caused heavy economic losses of Australia poultry in 1980s. There were also few case reports in China.Avian HEV, which belonged to the Hepeviridae family, was first isolated and characterized from chickens with HS syndrome in USA. Compared with mammalian HEV complete genomic sequences, avian HEV showed only 50% identities with mammalian HEV. But avian HEV is antigenically and genetically related with swine and human HEV. The full length of avian HEV genome is 6.6kb and is 600bp shorter than the length of mammalian HEV complete genome. The complete genome of avian HEV contains three open reading frames (ORF), ORF1, 2 and 3, besides the 5’cap and 3’PolyA structure. The ORF2 gene encodes the capsid protein and contains primary epitopes of avian HEV. Because there was no effective cell culture system for avian HEV, many researches focused on the ORF2 protein of virus substructure protein. Recently, the ORF2 protein of avian HEV expressed with prokaryotic and eukaryotic expression system has been characterized and was used to diagnosis for antibody test.1. Detection of avian HEV from China with RT-PCR method.Fecal, bile and serum samples were collected from chickens with HS syndrome or BLS disease in some fields, that there were high positive rate for avian HEV antibody according to the previous results of seroepidemiology investigated in our lab. Partial ORF2 gene of avian HEV was detected using RT-PCR method based on the primers described by other researchers. Partial ORF2 gene sequences was obtained from a flock of 35 or 37-week-old broiler breeder hens with HS syndrome. Meanwhile, sequences analysis revealed that the obtained sequences showed the highest identity with European isolates and phylogenetic trees showed that the isolates in this study are closely related to the Europe isolates and belonged to avian HEV genotype 3. 2. Analysis of the complete genome of avian HEV from China.In the study, twelve primers pairs were designed according to the referenced sequences in GenBank. Then six overlapping fragments were amplifying from the bile samples to be positive for avian HEV RNA. The complete genome of Chinese avian HEV isolate was assembled by using the 6 overlapping fragments and designated CaHEV. The complete genomic and different region sequence analyses indicated that CaHEV shared the highest identity (98.3%) with European avian HEV isolate. Compared the ORF2 amino acid sequences with avian HEV each other, the ORF2 gene shared 98.3%–99.7% aa sequence identities with each other. It indicated that there was one serotype among avian HEV isolates. ORF1 of CaHEV contained most mutations compared with prototype avian HEV. Phylogenetic trees of the full-length sequence of avian and mammalian HEV strains showed that avian HEV was segregated into a distinct branch separate from mammalian HEV and CaHEV belongs to avian HEV genotype 3.3. Pathogenesis research of avian HEV from China.Avian HEV stock of bile and fecal samples suspension were collected from challenged chickens inoculated with bile samples positive for avian HEV RNA detected with RT-PCR. Then the avian HEV stock was filtered and quantitated using GE method. In the study, eighty-four chickens of 15 week ages were divided into 3 groups which were separately housed. Chickens in two groups were inoculated with CaHEV stock by intravenously and orally, respectively. The third group was control group. Fecal samples were collected from the challenged chickens every one day post inoculation and serum samples were collected from every three days post inoculation. Meanwhile, two chickens were necropsied every one week post inoculation and the remaining chickens were necropsied in 12 wpi. Bile and live, spleen tissues were collected. The detection of all collected samples for avian HEV RNA using RT-PCR indicated that avian HEV RNA was first detected from fecal samples of challenged chickens in 3 dpi and was still detected in 54 dpi in intravenously group. Viremia in this group was first detected in 6 dpi and persisted to 21 dpi. In orally group, avian HEV RNA in fecal samples was first detected in 6 dpi and still in 62 dpi and viremia was first appeared in 12 dpi and disappeared in 33 dpi. Analysis of seroconversion to avian HEV antibody using indirect ELISA showed that the antibody titers of intravenously group wane rapidly when it reached the peak, but the antibody titers of orally group declined slowly when reached peak. In addition, partial chickens showed big live and spleen and hemorrhage of the liver and spleen in the two challenged group. Meanwhile, lymphocytic periphlebitis and phlebitis foci were observed in the liver sections from two challenged group. These results further confirmed the relationship between avian HEV and HS syndrome.4. Development and application of indirect ELISA for the detection of avian HEV antibody.An indirect ELISA was established for the detection of avian HEV antibody using the coated antigen of avian HEV truncated ORF2 protein through optimizing the different conditions. We investigated epidemiology of 11 chicken farms in Shandong province for avian HEV infection using the indirect ELISA. It was shown that avian HEV antibodies were detected in all 11 flocks. But the positive rate for avian HEV antibody among 11 flocks was significant difference. The positive rate reached 77% in two flocks. There were 493 of positive serum for avian HEV antibody in 1379 serum collected from the 11 flocks. It was suggested that avian HEV infection has been very prevalence in Shandong province.In addition, we also detected the serum against antibody from different aged chickens in five flocks of same chicken farm using the indirect ELISA. It was shown that the serum from 12 week-old-age chickens were positive for avian HEV antibody and the positive rate reached the largest in the serum from 20-30 week-old-aged chickens in all five flocks. In a flock, the positive rate reached the largest again in the serum of 40-50 week-old-aged chickens. Only 10% of chickens older than 60 weeks were seropositive. These results suggested that the chickens from five flocks were naturally infected by avian HEV in younger than 10 weeks according to the results of the serum from 11 or 12 weeks to be positive and the antibodies to avian HEV of mostly chickens older than 60 weeks disappeared.5. Seroconversion to avian HEV antibodies against different protein in inoculated chickens.In previous study, it was documented that primary epitopes located in the capsid protein of avian HEV and six antigen domains located in the 22 to 83aa, 339-383aa, 389-410aa, 461-492aa, 556-566aa and 583-600aa, respectively. In addition, ORF3 protein of avian HEV can induce immune responses in chickens. Then to investigate the difference of seroconversion to avian HEV antibodies against the different domains located in ORF2 protein and ORF3 protein, the serum from challenged chickens of different dpi were detected with indirect ELISA using different coated antigen including ORF2-F (22-83aa),ORF2-S(339-606aa),ORF2-5(339-442aa),ORF2-8(383-515aa),ORF2-9(412-546aa)and ORF3 protein. The results showed that the antibodies against ORF2-S were first detected in the serum from challenged chickens of 12 dpi and antibodies against ORF2-F and ORF3 was first detected in 15 dpi serum. But the titers of antibodies against ORF2-F and ORF3 wane rapidly and the titers of ORF2-S declined slowly when they reached the peak, respectively. It was suggested that antibodies responses to the antigen domain of 22 to 83aa located in the forward fragment of ORF2 protein and to ORF3 protein were transient and antibody responses to the latter fragment of ORF2 protein. The titers of antibody against ORF2-5 (339-442aa) in the serum of different dpi were similar as the titers of ORF2-S (339-606aa) detected with indirect ELISA. It was suggested the antigen domains located in 339 to 442aa primarily induced immune responses in the challenged chickens. In addition, according to the titers of antibody against ORF2-8 (383-515aa), we concluded that the 339 to 383aa region was the primary antigen domain in the latter fragment of ORF2 protein induced immune responses in inoculated chickens. Meanwhile, there were no antibody responses to the 412 to 546aa region of ORF2 protein in inoculated chickens because of no reaction between serums of different dpi with ORF2 protein using indirect ELISA.6. Self-assembly of empty virus-like particles of avian HEVThree truncated proteins including complete ORF2 protein, deleted 56 amino acids in N terminal (ORF2Δ56) and C-terminal 268 amino acids (ORF2-268) were expressed with baculovirus system. Three truncated ORF2 proteins were successfully expressed in baculovirus system through identification of IFA and Western-botting methods. Meanwhile, it was found that the ORF2Δ56 protein was secreted into the cell culture medium when it was expressed with baculovirus system in sf9 cells. Compared with the proteins of ORF2 and ORF2Δ56 expressed with E.coil system, it was shown some proteins to be size smaller than expected protein were produced in baculovirus system. The reason is that the protein expressed with baculovirus may be proteolytic processing in the sf9 cells. The empty virus-like particles of avian HEV was examined with EM when the protein secreted in cell culture medium expressed with baculovirus for ORF2Δ56 protein was purified.更多还原