【作者】 张兴晓；

【导师】 柴同杰； 姜世金；

【作者基本信息】 山东农业大学 ， 预防兽医学， 2009， 博士

【摘要】 鸭圆环病毒(Duck circovirus,DuCV)是圆环病毒科(Circoviridae)圆环病毒属(Circovirus)的一新成员,2007年ICTV(The International Committee on Taxonomy of Viruses)将DuCV列入圆环病毒属中的暂定种。DuCV感染可导致鸭子羽毛凌乱、生长迟缓、体重减轻等症状。本文拟从DuCV的流行病学调查、克隆测序及分子遗传变异、人工感染的病理组织动态变化、建立血清学检测方法和体外组织细胞培养等方面进行研究和探索。1、山东省樱桃谷鸭群中鸭圆环病毒及混合感染的流行病学调查本研究旨在调查中国山东省樱桃谷鸭(Cherry Valley ducks)群DuCV的感染与流行情况。2006-2007年间,从山东省70个樱桃谷肉鸭养殖场随机选取742只鸭,采用PCR和Dot-blot方法,对DuCV感染情况进行流行病学调查。结果显示,被调查鸭中有33.29%感染了DuCV;3、4周龄的鸭子较2周龄的更易感;PCR鉴定方法比Dot-blot敏感。此外,与DuCV阴性鸭比较,DuCV阳性鸭与鸭1型肝炎(25.50%vs7.47%)、鸭疫里默氏菌(23.48%vs8.28%)及大肠杆菌(16.19%vs4.85%)的混合感染率较高。调查显示,DuCV在樱桃谷鸭群中普遍存在,且DuCV呈阳性的鸭更易与其它病原发生混合感染。2、鸭圆环病毒的克隆测序及序列分析从中国大陆樱桃谷鸭中得到LY0701、WF0701两株DuCV的全基因组,Genbank登录号分别为EU022374,EU022375。基因序列特征分析表明,这两株病毒的基因组均具有以下分子特征:(1)颈环结构;(2)滚环复制;(3)四个重复序列;(4)两个主要开放阅读框,分别编码复制蛋白Rep基因和编码衣壳蛋白Cap基因。通过与已发表的序列比较分析,所分离的两株病毒分别在两个分支上,LY0701株与台湾T1、T2、T3株、中国LY0701同源性高达96.7%以上,位于一个分支上,WF0701株则与美国株、德国株以及台湾的T4株更为接近,同源性达到93.5%以上,位于另一分支上。3、樱桃谷鸭人工感染鸭圆环病毒的病理动态变化本研究以已经完成全基因组序列测定的LY0701株DuCV为研究材料,利用感染LY0701株的阳性病料人工接种DuCV阴性的3周龄健康雏鸭,分别于攻毒后第2、4、6、8周对发病鸭进行病理观察。结果表明,与对照组相比,LY0701株阳性病料感染的雏鸭临床症状均出现发育不良,消瘦,羽毛凌乱等现象。剖检显示胸腺、法氏囊、脾脏、肝脏不同程度出血、肿胀,其它内脏器官病变不明显。病理切片显示胸腺、法氏囊、脾脏、肝脏出血,淋巴细胞坏死、崩解等变化。4、DuCV Cap蛋白基因原核表达及产物免疫原性研究根据已发表的DuCV LY0701株Cap基因序列,设计一对引物,以测序正确的LY0701株Cap基因质粒为模板,扩增出相应的基因片段,以pGEX-6P-1为载体进行连接,转化大肠杆菌BL21株,挑选阳性克隆,测序验证正确后,分别进行原核表达。SDS-PAGE电泳表明,表达产物的条带大小与理论值相符。利用原核表达产物免疫BALB/c小鼠,制备抗Cap蛋白的血清,标记为Ab-Cap。以FITC标记的羊抗鼠IgG为二抗,建立了间接免疫荧光染色法(IFA)。利用Ab-Cap对人工感染病料的检测表明,在脾脏、法氏囊、胸腺、肝脏的冷冻切片中可检测到被荧光染色的完整的细胞;在心脏、肺脏、肾脏等病料中未检测到荧光染色的阳性细胞。为进一步确定IFA检测的敏感性,临床上分别收集疑似DuCV感染的96只鸭子的胸腺、法氏囊、脾脏、肝脏、骨髓,利用IFA进行DuCV检测。结果表明,当鸭子感染DuCV后,脾脏的病毒检出率最高,达到18.75%,其它脏器的病毒检出率从高到低依次为法氏囊、肝脏、胸腺。利用Ab-Cap对临床病例检测表明,该方法更适合用于8周龄以上鸭子的脾脏检测。5、DuCV在不同组织及细胞中培养的探讨对PCR检测DuCV强阳性的5份病料(LQ76,LQ66,LQ14,LY5,WS23)进行处理并(1)通过鸭胚的卵黄囊、绒毛尿囊膜、尿囊腔等组织进行接种,(2)鸭胚成纤维细胞、鸭胚肝细胞、鸭胚肾细胞、鸭胚脾细胞接种阳病料组织,将收获的组织和细胞培养物连续盲传10代,将各代次培养物利用PCR、电镜、间接免疫荧光染色法(IFA)进行鉴定。结果表明,利用上述组织和细胞对鸭圆环病毒进行培养,未能获得成功。更多还原

【Abstract】 Duck circovirus (DuCV) is listed as a tentative member of the Genus circovirus by the most recent 8th ICTV Report. Ducks infected by DuCV exhibit disordered feather, retarded growth, reduced body weight. Pathological examination of the sick ducks revealed necrosis in lymphoreticular tissue, reduction in the lymphocyte count or proliferation of lymphoid tissue. Circovirus infection is characterized by lesion in lymphoid tissue that leads to the reduction or suppression in immunity (Soike et al., 2004) and secondary infection. Coinfection by DuCV and other viral or bacterial pathogens is very frequently examined. In the present study, we conducted and discussed epidemiological investigation, molecular gentic variation, Preliminary Pathologic Observations, serological methods and culturing in vitro.1、An epidemiological investigation on duck circovirus in Shandong, ChinaCoinfection of duck circovirus (DuCV), Riemerella anatipestifer (RA), Escherichia coli (E. coli) and duck hepatitis virus I (DHV-I) in Cherry Valley ducks in Shandong, China was investigated by PCR-based methods. 742 ducks sampled during 2006-2007 from 70 duck farms were examined by PCR and dot blot analysis. Overall DuCV-infection rate was 33.29%. Ducks at 3-4 weeks of age were more susceptible to DuCV infection, comparing with those at 2 weeks of age. The DuCV-positive ducks had a higher rate of infection by duck hepatitis virus I (25.5% vs 7.475), RA (23.48% vs 8.28%) and E. coli, comparing with DuCV-negative ones. This investigation showed that DuCV infection is common in Cherry Valley ducks.2、Sequence Analysis for the Complete Genome of Duck Circovirus IsolatesThe complete genome of two strain Duck circovirus (DuCV), LY0701 and WF0701, were gained from cherry valley ducks in mainland of China for the first time. The numbers of Genbank accession database are EU022374 and EU022375, respectively. Sequences analysis shows that the two stains DuCV exhibited the four genetic features characteristic of other circovirus: a stem-loop structure; four intergenic repeats; the presence of the ORFs rep and cap; the conserved motifs for the rolling circle replication and for the dNTP binding domain observed in the Rep protein. Comparisons of the two strains with that of representative DuCV strains demonstrated that the two DuCV strains were grouped in two main branches. LY0701, including the DuCV T1/2002, T2/2002, T3/2002 from Taiwan of China and FJ0601 from mainland of China were in one branch. The identity was as high as 96.7%. WF0701, including the German DuCV, the USA DuCV, and the DuCV T4/2002 from Taiwan were another branch. The results of the study showed the variety of DuCV in China.3、Preliminary Pathologic Observations of Cherry Valley Duck Artificially Infected with Duck CircovirusThe whole genome of duck circovirus (DuCV)was obtained from Cherry Valley ducks from a diseased duck farm located in China’s Shandong Province and named LY0701 strain, with its Genbank login no. EU022374. Artificially inoculate DuCV negative duckling at three weeks of age by using positive sample infected with LY0701strain, and conduct pathological observation separately after 2, 4, 6, 8 weeks. Compared with the control group, the clinical symptoms of the ducklings infected with positive LY0701 all showed stunted, thin, messy feathers, and so on, autopsy showed that there was bleeding and swell of different degrees in thymus, bursa, spleen and liver, other internal organs with unobvious lesions. Pathological section showed bleeding in thymus, bursa, spleen and liver, lymphocyte necrosis and/or collapse.4、Study on DuCV Cap Gene Expression Products and ImmunogenicityAccording to the published sequence of Cap gene of (DuCV) LY0701 strains, design a pair of primers for the each gene, amplify the corresponding gene fragment based on the plasmid gene of Cap of DuCV LY0701 strains of correct sequence as a template, then connect respectively taking pGEX-6P-1 as the carrier, transform coli strain BL21 and clone the positive ones, finally carry out prokaryotic expression when the sequence is correct and verified. The SDS-PAGE electrophoresis shows that the band of expression products meets the theoretical values in size. To immunize the BALB / c mouse respectively by using the prokaryotic expression products, and prepare the serum of anti-Cap, marked as Ab-Cap. Establish the indirect immunofluorescence staining assay (IFA) by using goat anti-mouse IgG-FITC as secondary antibody. The results of Ab-Cap assay for diseased samples of artificial infection show that the entire staining cells can be detected in frozen sections of spleen, bursa, thymus, and liver. But the Ab-Cap has not detected the typically staining cells in samples of heart, lung, and kidney.In order to further determine the phagocytosis of DuCV on organs and tissues, bursa, spleen, liver, and bone marrow of 96 suspected DuCV infected ducks were collected and examined using IFA, the results showed that the detection rate of spleen of DuCV infected ducks was the highest, up to 18.75%, followed by Bursa, thymus, liver.5、Study on Culture of DuCV In Different tissues and cells5 samples (LQ76,LQ66,LQ14,LY5,WS23)were inoculated by different ways: Duck embryonal yolk cavity, allantocherion, allantoic cavity, embryonal body, duck embryo fibroblast, hepatic cell, renal cells, spleen cell. Tissue and cell cultures were continued 10 generations by blind passage. All cultures were identified by PCR, electron microscope, IFA. The results showed that all the above-mentioned methods were not success.更多还原