【作者】 朱丹；

【导师】 谢鹏；

【作者基本信息】 重庆医科大学 ， 神经病学， 2009， 博士

【摘要】 背景当中国的SARS、马来西亚的尼巴病毒(NiV)、美国的西尼罗河病毒(WNV)以及全球的禽流感病毒所引起的一场场席卷全球的各种病毒性疾病爆发流行时,人们在极度恐慌的同时也进行了反思,为什么不能预测这些传染病的爆发流行?为什么对疾病的流行缺乏有效的监控措施和疫情预报?现有的防治策略和措施为什么收效甚微?这些都促使我们对新发病毒性疾病的防治工作和公共卫生体系的建设有了新的认识。及早的对可能成为的新发感染性疾病致病原的病毒给予关注,尽早的研究发现这些病毒的起源、传播、与临床表现和预后相关的宿主因素、诊断方法和治疗措施,为防止这些病毒的大流行打下基础,已成为目前迫切需要解决的重大公共卫生问题。目的本研究旨在关注中枢神经新发感染性病毒Borna病病毒(Borna disease virus,BDV),拟对BDV一步法实时RT-PCR检测方法进行一些有益的探索,并和前期开发建立的BDV荧光定量套式逆转录酶聚合酶链反应(FQ-nRT-PCR)检测方法进行比较,优选出快速、准确、敏感、特异的检测BDV的方法,为大规模流行病学监测提供一种简便、可靠的检测手段。同时,应用所建立的检测方法,对采集自中国新疆伊犁地区的和重庆三峡库区的马、猪外周血、脑组织样本分别进行BDV检测,以获得我国西部部分地区的BDV的病毒流行病学资料,为BDV感染性疾病的可能爆发流行提供预警,同时也为某些神经精神疾病的病因学诊断及其防治提供新的思路和依据。方法1根据GenBank提供的基因序列,针对BDV基因的保守区域(BDV p24基因),设计并合成特异性引物和荧光标记TaqMan探针。从含有目的基因的质粒出发,利用PCR方法扩增获得目的基因,克隆至pBS-T载体进行体外转录,将得到的RNA纯化并使用RiboGreen方法进行定量后作为阳性标准品,初步建立BDV的一步法实时RT-PCR方法,对其敏感性和特异性进行探讨;同时与前期本课题组建立检测BDV的FQ-nRT-PCR方法进行比较分析。2自2007年6月起,采集自中国西部部分地区动物标本共480份(新疆伊犁地区天然牧场上放养马的外周血和脑组织标本120份、重庆及三峡库区乡村饲养的猪外周血标本360份),使用密度梯度离心法分离外周血淋巴细胞,应用Trizol法提取细胞总RNA。用优选的BDV检测方法,对样本进行BDV检测。对阳性样本进行PCR产物序列鉴定、序列分析等研究,在可能的情况下进行病毒分离培养,并提交流行病学调查报告。结果1对所设计的BDV引物和探针序列,登录美国国立卫生研究院网站(http://www.ncbi.nlm.nih.gov/BLAST/)进行Blast检索,所设计的引物和探针可以与已公布的BDV病毒株序列匹配。2本研究建立BDV一步法实时RT-PCR方法的最低检出限即敏感性为1.7×102copies/μl,标准曲线的定量范围分别为1.7×102～1.7×106copies/μl,相关系数R2分别为0.9998,不与其他病毒发生非特异反应。该方法具有较好的灵敏性和特异性,在BDV流行病学研究中可以选择使用。3对采集自中国新疆伊犁地区的和重庆及三峡库区的马、猪外周血、脑组织样本分别进行BDV检测,新疆伊犁地区有3例马外周血和脑组织中同时检测到博尔纳病毒,阳性率为2.5%(3/120)。扩增产物序列与其他国外马源BDV分离株同源性大于93%,与标准株He/80同源性达到98%以上。重庆及三峡库区的BDV p24基因片段阳性率在360只猪的PBMC BDV-p24基因的阳性检出率为4.44%。序列分析结果表明,重庆及三峡库区猪感染的BDV核苷酸序列与马He/80同源性为100%,并且编码的氨基酸序列也相同。结论1本研究构建了BDV p24基因检测用的RNA及DNA标准品,并初步建立了BDV的一步法实时RT-PCR检测方法,与本课题组前期建立的FQ-nRT-PCR检测方法相比,FQ-nRT-PCR方法,具有更好的灵敏性和特异性,并且有稳定性和重复性好等特点,适于流行病学研究和BDV相关性疾病的诊断。2初步流行病学研究提示我国西部新疆伊犁地区马群中存在BDV的自然感染。该地区BDV流行株与标准株He/80存在高度的同源性。3初步流行病学研究提示我国重庆及三峡库区猪中存在BDV的自然感染。该地区感染的BDVP24核苷酸序列与He/80株具有高度同源性。更多还原

【Abstract】 BackgroundThe quick spreading of infectious viruses, including SARS in China,Niv in Malaysia, West nile virus in United States, and Avian Influenza Virus worldwide, have brought great panic to mankind. People started to reflect the reason why all those epidemics can’t be predicted, monitored and stopped effectively. Early detection of potential outbreaks, exploration of origin, spreading, clinical manifestations- and- prognosis- associated host factors, diagnosis and clinic treatment, might be the best way to prevent epidemics.ObjectiveThe objective was to establish one-step real-time RT PCR, which is superior to traditional Fluorescent quantitative nested RT PCR, to rapidly detect Borna disease virus in diseased hosts, for nationwide molecular screening of potential infections. In addition, we also aim to perform molecular epidemiological study of BDV infection in brain tissues and blood samples of horses and swine in Xinjiang and Chongqing.Methods1. Based on GenBank target sequences, we designed TaqMan probes specifically targeting the conservative region of BDV p24 and established a one-step RT-PCR for detecting BDV infections. The sensitivity and specificity of established one-step RT-PCR were tested and compared with FQ-nRT-PCR.2. Since June 2007, a total of 480 samples collected from animal species in Western China were centrifuged to separate peripheral blood mononuclear cells (PBMCs). Total RNA was extracted by using Trizol agent. We have detected all the samples by using the optimized PCR assays. All positive samples were sequenced and further virus isolation was performed for epidemiological purpose.Results1. We logged on the NIH website (http://www.ncbi.nlm.nih.gov/BLAST/) to perform a BLAST search for the primers and probes. The designed primers and probes are matched for the published BDV sequences.2. The detection limit of the current one step RT-PCR was 1.7×102copies/μl,with a target range of 1.7×102～1.7×106copies/μl. The assay is highly specific and no cross-react with other virus sequences. The method can be properly applied to the BDV epidemiology research, with good sensitivity and specificity.3.We examined the PBMCs and brain tissues from horses and swine in Xinjiang and Chongqing for possible BDV infections. BDV was detected from both PBMCs and brain tissues in three horses (2.5%) in Yili, Xingjiang. The amplified positive products were > 98% homologous to standard He/80 strains. Of the 360 swine samples tested, BDV p24 RNA positive rate was 4.44% in Chongqing. The amplified positive products were 100% homologous to standard He/80 strains. Conclusion1. We have established one step RT-PCR and FQ-nRT-PCR for detection of BDV p24 RNA in diseased hosts. Compared with the one step RT-PCR, FQ-nRT-PCR is more sensitive and specific for epidemiological screening of potential BDV infections.2. The results of our epidemiological study suggest that there is BDV natural infection in horses in Yili, Xinjiang province. The BDV strains are highly homologous to He/80 standard strains.3. The results of our epidemiological study also suggest that there is BDV natural infection in swine in Three Gorges Regions, Chongqing. The BDV strains are highly homologous to He/80 standard strains.更多还原