【作者】 展群岭；

【导师】 谢鹏；

【作者基本信息】 重庆医科大学 ， 神经病学， 2010， 博士

【副题名】样本采集病毒检测种系发生分析细胞模型建立试剂盒评价

【摘要】 背景WHO公布2003年席卷全球的SARS爆发流行,涉及32个国家和地区,全球累计病例8 422例,死亡人数919人,病死率约11%。2004年禽流感病毒甲型H5N1在南亚和东南亚爆发流行,致使超过1亿只家禽被捕杀,并传播人类导致感染患者的死亡。2009年4月自墨西哥出现的第一例甲型H1N1流感病毒感染患者至2010年3月,该病毒的大流行已蔓延全球213个国家和地区。2010年4月,WHO公布迄今为止甲型H1N1病毒大流行已造成17 000余人死亡。截至2010年3月31日,中国31个省累计报告甲型H1N1流感确诊病例120 000例,死亡病例超过800例。近7年新发感染性疾病(emerging infectious disease,EID)事件触目惊心,对人类健康、经济发展和社会稳定产生了巨大的影响,使人们对EID积极防控和健全相关公共卫生安全体系产生了许多新的认识,并对EID病原体给予了高度关注。目的本研究通过BDV分子流行病学调查和研究,建立中国西北伊犁地区新发病毒BDV感染动物样本库,获得BDV流行病学资料,了解不同种属动物BDV流行现状,分析BDV可能的种系来源,为制定预防和控制BDV爆发流行预案提供可靠依据,为健全公共卫生安全体系奠定坚实的基础。同时,建立BDV p40-OL细胞模型,为BDV发病机制的蛋白质组学研究提供平台;进一步评价BDV荧光定量PCR诊断试剂盒,为BDV流行病学调查和临床研究提供可靠的检测手段。方法1.根据BDV感染宿主地源特殊性和种属多样性,选择中国西北新疆伊犁地区作为样本采集地点;伊犁马、新疆驴、新疆双峰驼、天山马鹿和不同品种犬作为样本采集动物;外周血和脑组织作为样本采集对象。2.采用FQ-nRT-PCR方法,检测8个品种150例犬PBMCs BDV p24 RNA基因片段。用检测BDV p40 RNA片段和质粒pMD19 BDV H1766 p24标准品验证样本BDV p24阳性产物,排除可能出现的假阳性。验证后的阳性产物进行基因测序、核苷酸和氨基酸序列同源比对以及系统发生学分析。3.在5个种属8个品种873例动物PBMCs中,对伊犁马、新疆驴和哈萨克牧羊犬共检出的19例BDV p24 RNA阳性片段,进行BDV标准株He/80、strain V和H1766比对,分析核苷酸和氨基酸同源相似度,重构基因系统发生树,分析BDV可能的种系来源。4.培养人OL细胞株细胞,用含有BDV p40基因的质粒pEGFP- N1- BDV p40,经脂质体转染OL细胞。通过G418筛选已转染的OL细胞,获得BDV p40蛋白稳定表达的转染态OL细胞。RT-PCR扩增,琼脂糖凝胶电泳鉴定质粒BDV p40基因表达;观察GFP表达、测定IHC和ELISA,鉴定BDV p40蛋白表达。最后,建立稳定表达BDV p40的转染态OL细胞模型。5.试剂盒评价在热稳定性评价方面,将BDV p24荧光定量PCR诊断试剂盒中试剂分装多份一次PCR扩增反应量。随机取出其中2份分别置于25℃和37℃两种温度的恒温水浴箱内进行热处理。处理时间分别选择3 h、6 h、9 h、12 h、24 h、48 h和96 h共7个时间段。在相应温度和热处理时间完成后,对6个浓度梯度的质粒pMD19 BDV H1766 p24标准品进行PCR扩增,扩增产物用琼脂糖凝胶电泳鉴定。通过对PCR反应的CT值和R2值比较,评价试剂盒的热稳定性。在BDV检测信度评价方面,选择定量BDV(+)OL细胞和质粒pMD19 BDV H1766 p24标准品,进行BDV p24基因片段PCR扩增。通过比较两种检测对象检出BDV p24浓度梯度的一致性,评价试剂盒病毒检测结果信度。结果1.在样本采集方面,伊犁地区样本采集动物归属于5个种属8个品种。种属包括伊犁马、新疆驴、新疆双峰驼、天山马鹿和犬;品种有哈萨克牧羊犬、德国牧羊犬、卡罗来纳犬、西伯利亚雪橇犬、波音达猎犬、比格猎犬、德国杜宾犬和斗牛梗犬。样本采集动物数量合计897例,其中伊犁马582例,新疆驴204例,新疆双峰驼4例,天山马鹿1例,哈萨克牧羊犬95例,德国牧羊犬28例,卡罗来纳犬14例,西伯利亚雪橇犬8例,波音达猎犬6例,比格猎犬4例,德国杜宾犬3例,斗牛梗犬2例。样本采集对象为外周血、脑组织和全脑。采集样本的数量为外周血样本897份、脑组织样本1573份和全脑样本43份。2.在病毒检测方面,在8个品种150例犬中,仅哈萨克牧羊犬检出BDV p24片段,阳性率为11.0%(10/91)。基因序列与德国马BDV He/80和德国绵羊BDV S6病毒株同源相似度分别为99.2%和95.7%,氨基酸相似度为100%和89.3%。亲缘关系与He/80最近,其次为S6。3.在种系发生学方面,19例BDV p24核苷酸序列一部分形成伊犁独立支系,其余部分汇聚至伊犁-德国-瑞士混合支系。独立支系与标准病毒株无聚合,核苷酸序列同源相似度为98%,氨基酸序列为100%;德国马He/80汇聚混合支系,核苷酸与氨基酸序列与He/80同源相似度均为100%。4.在细胞模型建立方面,用RT-PCR扩增经pEGFP-N1-BDV p40转染的OL细胞中BDV p40基因核苷酸序列片段,扩增产物在琼脂糖凝胶电泳154 bp处出现一条清晰的条带,其大小与目的片段一致。倒置荧光显微镜观察,OL细胞核GFP表达量明显大于细胞质(细胞核/细胞质=2.43),证明了BDV p40蛋白核定位的生物学特点。用BDV多克隆抗体和CIC单克隆抗体检测pEGFP-N1-BDV p40、pEGFP-N1和正常OL细胞中BDV p40蛋白表达,结果显示,前者均为阴性,后者均为弱阳性,三者之间无差异。5.在试剂盒评价方面,试剂盒热稳定评价显示,经25℃和37℃热处理后,各温度7个不同时间段BDV p24 PCR反应CT值之间无差异,保持正常PCR扩增效率的热处理持续时间范围为3 h～96 h;25℃和37℃两种温度条件下不同时间段平均R2值之间亦无差异。BDV检测信度评价方面显示,在定量BDV(+)OL细胞(12个浓度梯度即100～10-11)和质粒pMD19 BDV H1766 p24标准品(6个浓度梯度即100～10-5)中,均可检测4个浓度梯度即100、10-1、10-2和10-3,最低检测浓度为10-3。结论1.伊犁地区所采集不同种属和品种的动物血液和脑组织样本量,可基本反映该地区BDV自然感染现状,纳入动物性别和年龄的同质性进一步提高了调查结果的可靠性。在国内外首次建立了马科动物快速开颅全脑采集的新方法。2.伊犁地区可能存在BDV自然感染,哈萨克牧羊犬为BDV贮存宿主,其自然感染阳性率为11.0%;该地区BDV流行病毒株可能与伊犁马BDV He/80的交叉感染和引进德国Saxony美利奴绵羊BDV S6病毒株的输入感染有关。3.首次提出了新疆伊犁河谷动物宿主中可能存在地源性BDV独立种系,形成哈萨克牧羊犬BDV KT078和KT079病毒株;同一种系BDV株在伊犁河谷不同种属动物间的感染,可能源于外来疫病病原体经草原丝绸之路的传播。4.质粒pEGFP-N1-BDV p40转染人OL细胞后BDV p40基因和蛋白在细胞内的稳定表达,建立了转染态BDV p40-OL细胞模型,为探索BDV p40单一病毒蛋白在宿主中的作用和BDV致抑郁症发病机制的蛋白质组学研究提供了有效的手段。5.荧光定量BDV p24诊断试剂盒在一定范围内不受温度(25℃/ 37℃)和在该温度下持续使用时间(3 h～96 h)的限制,具有较好的热稳定性;该试剂盒具有极高的BDV检测信度,可检测宿主细胞中10-3 BDV浓度。更多还原

【Abstract】 BACKGROUNDWHO announced that an outbreak of SARS involving 32 countries and regions over the world in 2003 led to 8 422 infected cases including 919 deaths, and the case fatality rate was nearly 11%. The outbreak of influenza A H5N1 of the bird flu virus in South and Southeast Asia in 2004 resulted in more than 100 million chichens killed and the virus even spread to humans to cause the infected patients dead. In April 2010, WHO has published that more than 213 countries and territories worldwide have reported laboratory confirmed cases of pandemic influenza A H1N1 2009, including over 17 000 deaths from the first case of influenza A H1N1 in Mexico in April 2009 to March 2010. As of March 31, 2010, 120 000 confirmed cases with influenza A H1N1 had been cumulatively reported by 31 provinces in China and the deaths were beyond 800 cases. The shocking EID (emerging infectious disease, EID) events have hugely impacted human health, economic development and social stability for the past 7 years, which make people produce more new views on active prevention and control of EID and perfection of related public health, and pay high attention to the pathogen of EID.OBJECTIVEThe purposes of this study are to establish sample database of animals infected by EID BDV in northwest of China,Ili,Xinjinang,to obtain epidemiological BDV data, to come to understand the epidemic status of different species of animals,and to analyze the possible phylogeny of BDV, which provide reliable data for making preplans of prevention and control of BDV outbreak and lay solid foundation for improving public health security system. At the same time, the cell model of BDV p40 viral protein transfected will be constructed to establish the research platform for BDV pathogenesis. The BDV p40 fluorescent quantitative PCR diagnosis kit will be further evaluated to provide a reliable means of detection of BDV epidemiological investigation and clinical research.METHODS1. In sample collection, according to the specificity of geographical origins and species diversities of the hosts infected by BDV, Ili region in Xinjiang was selected as the point of sampling collection; Ili horse, Xinjiang donkey and dogs of different breeds as sampling animals; peripheral blood and brain tissue of animals as sampling objects.2. In virus detection, the gene fragments of BDV p24 RNA were detected from PBMCs in 8 breeds of 150 dog’s samples with using FQ-nRT-RT PCR. The possible false positives were excluded by determination of both BDV p40 RNA fragments and the plasmid standard of pMD19 BDV H1766 p24. The analysis was performed on genetic sequence, homologous comparison, amino acid sequence and phylogeny after BDV p24 positive products were validated.3. In phylogenetic analysis, the 19 nucleotide sequences of BDV p24 positive fragments of Ili horses, Xinjiang donkeys and Kazakh tobets (a Kazakh shepherd dog) had been detected with using FQ-nRT-RT PCR from PBMCs in 873 animals of 5 species and 8 breeds. The homologous similarity of nucleotide and amino acid sequences was analyzed by alignment of BDV standard strain He/80, strain V and H1766, and gene phylogenetic tree was reconstructed to analyze BDV phylogeny.4. In cell model construction, the cell of human oligodendrocyte strain (OL cell) was cultured, which had been established from human oligodendroglioma cell. OL cells were transfected by plasmid pEGFP-N1-BDV p40 with BDV p40 gene by liposome (lipofectamine- 2000). Having been repeatedly screened by G418, the OL cell turned to be a transfected one which may stably express the BDV p40 protein. The expression of the BDV p40 gene of plasmid in the OL cells was identified by the techniques of RT-PCR amplification and agarose gel electrophoresis while the expression of BDV p40 protein by the observation of green fluorescent protein expression, IHC and ELISA determination. The model of transfected type of OL cell which may stably expressed BDV p40 protein was established.5. Kit evaluation. The first, in the evaluation of thermal stability, the reagent in the kit was divided into volume units of one PCR amplification reaction, and then two units were randomly selected to get heat treatment by 25℃and 37℃in constant temperature water bath, respectively. The seven durations of heat treatment were chosen including 3 h, 6 h, 9 h, 12 h, 24 h, 48 h and 96 h. The plasmid standard of pMD19 BDV H1766 p24 in 6 concentration gradients was immediately amplified with using PCR after accomplishment of heat treatment in the corresponding temperature and time. PCR products were identified by agarose gel electrophoresis and the kit thermal stability was evaluated by comparison of the CT and R2 values of PCR reaction in different temperatures and time durations. The second, in the reliability evaluation of BDV detection, the BDV p24 gene fragment in the quantitative BDV (+) OL cell and in the pMD19 BDV H1766 p24 plasmid standard were selected to be amplified by PCR, respectively. The kit reliability was evaluated by comparison of consistency of concentration gradients detected from the two detective objects.RESULTS1. In sample collection, the samples in Ili region belonged to 12 different species and breeds, including Ili horse, Xinjiang donkey, Xinjiang Bactrian camel, Tianshan red deer, Kazakh Tobet, German shepherd, Carolina dog, Siberian husky, Pointer hound dog, Beagle dog, German Doberman dog and Pit bull terrier. The total numbers of sampling animals was 897 cases, of which Ili horses were 582, Xinjiang donkey 204, Xinjiang Bactrian camel 4, Tianshan red deer 1, Kazakh Tobet 95, German shepherd 28, Carolina dog 14, Siberian husky 8, Pointer hound dog 6, Beagle dog 4, German Doberman dog 3 and Pit Bull Terrier 2. The sampling kinds were 3 in all, including peripheral blood, brain tissue and whole brain. The sampling numbers contained 897 blood samples, 1 573 of brain tissue and 43 of whole brain. The blood samples included those of 528 Ili horses, 204 Xinjiang donkeys, 160 different breeds of dogs, 4 Xinjiang Bactrian camels and 1 Tianshan Red Deer. The brain tissue samples consisted of those of 1 165 Ili horses (233 samples from frontal, parietal, occipital, temporal lobes or midbrain for each), of 305 Xinjiang donkeys (61 samples from frontal, parietal, occipital, temporal lobes or midbrain for each), and of 103 dogs (right temporal lobe). The whole brain samples involved in those of 37 Ili horse and 6 Xinjiang donkeys. 2. In virus detection, BDV p24 RNA fragments were found out only in Kazakh Tobet in 8 breeds of 150 cases and their overall positive rate was 11.0% (10/91). Compared with the strain BDV He/80 from horse and that of BDV S6 from sheep in Germany, the homologous similarity of Kazakh Tobet was 99.2% and 95.7%, and that of amino acid was 100% and 89.3%, respectively. The kinship of Kazakh Tobet was close to He/80 and next to S6.3.In phylogenetic analysis, one part of nineteen nucleotide sequences formed Ili independent branch and the other clustered within Ili - Germany - Switzerland mixed branch. The independent branch did not cluster with standard strains and the homologous similarity of nucleotide sequences was 98% and that of amino acid 100% in independent branch. The German horse He/80 clustered into mixed branch and the homologous similarity of both nucleotide and amino acid sequences was 100% aligned by He/80 in mixed branch.4. In cell model construction, the conservative fragment of nucleotide sequence of BDV p40 gene in transfected type of OL cell was amplified by RT-PCR. The amplified product appeared a clear 154 bp band in agarose gel electrophoresis, which of the size was consistent with the target sequence. The expression of green fluorescent protein in OL nucleus was significantly greater than that in the cytoplasm (the gray value of nucleus / cytoplasm = 2.43) under the Inverted fluorescence microscope, which was fully consistent with biological nuclear localization of BDV p40 protein. The expression of BDV p40 protein was determined by polyclonal antibody with cell immunohistochemistry and CIC monoclonal antibody with ELISA in pEGFP-N1-BDV p40, pEGFP-N1 and normal OL cell. The results showed that there were negative in the former while all weak positives in the latter in which there was no significant difference in three kinds of cells.5. In the evaluation of thermal stability, there was no difference among the CT values of PCR reaction in 7 different heat treatment durations for each temperature after the kit got to heat treatment by 25℃and 37℃.The range of duration to keep normal PCR amplification efficiency was from 3 h to 96 h. There was no difference between the mean R2 value of two different temperatures (25℃and 37℃). In the reliability evaluation of BDV detection, the four concentration gradients (100-10-3) were detected by the kit in the concentration gradients (100-10-11) of quantitative BDV (+) OL cell and of pMD19 BDV H1766 p24 plasmid standard (100-10-5). The lowest detective concentration was 10-3.CONCLUSION 1. The sample size of the blood and brain tissue collected in the Ili region in different animals could basically reflect the status of BDV natural infection in the region. The sex and age of enrolled animals had good homogeneity which further improved reliability of the survey results. A new method of rapid craniotomy for the whole brain of equine was established for the first time at home and abroad.2. There may exist BDV natural infection in Ili region, and Kazakh Tobet was the reservoir host which positive rate of natural infection was 11.0% (10/91). BDV endemic strains in the region were concerned with the cross-infection of Ili horse with He/80 and the importing infection of Merino sheep with BDV S6 introduced from Saxony, Germany.3. It was first put forward that there could exist BDV independent strain due to geographical origin in host animals in Ili valley, Xinjiang. The reason that different kinds of hosts were infected by the same strain virus was the spread of foreign pathogen via the Prairie Silk Road.4. The transfected cell model of BDV p40-OL was established through human OL cells transfected by Plasmid pEGFP-N1- BDV p40. The cell model could provide an effective means for exploring the function of single viral protein in the host cell and the proteomics research for pathogenesis of depression caused by BDV. 5. The kit was of good thermal stability for it was not restricted by a certain range of temperature (25℃/ 37℃) and of sustainable time (3 h-96 h) at the temperature. It was of very high reliability of BDV detection for it could detect 10-3 BDV concentration the in OL cell.更多还原