蝙蝠感染丝状病毒的血清学调查

发布时间：2018-01-13 00:36

本文关键词：蝙蝠感染丝状病毒的血清学调查　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：丝状病毒(Filovirus)属于单股负链RNA病毒目(Mononegavirales),目前包括三个属,分别为埃博拉病毒属(Ebolavirus)、马尔堡病毒属(Marburgvirus)和库瓦病毒属(Cuevavirus),前二个属主要分布于非洲,后一个属分布于欧洲。丝状病毒,对人具有高度的传染性和致死性,所以国际上将该科一些成员列为潜在的生物战剂,例如扎伊尔埃博拉病毒(Zaire Ebola virus,ZEBOV)和马尔堡病毒(Marburgvirus,MARV)。除了雷斯顿埃博拉病毒(Reston Ebolavirus,REBOV)的自然宿主是猪以外,其余已知的埃博拉病毒、马尔堡病毒以及库瓦病毒的自然宿主是蝙蝠。我们实验室于2015年首次在亚洲(中国云南省)发现了新型蝙蝠丝状病毒,说明亚洲地区的蝙蝠也有丝状病毒的分布,但其种类、感染率以及公共卫生意义尚不明确,而我国蝙蝠种类和数量繁多,因而建立一种快速、高效和准确的蝙蝠丝状病毒感染血清的检测方法并以此开展血清流行病学调查具有十分重要的科学意义。本研究为了建立检测MARV、REBOV、ZEBOV、库瓦病毒(Lloviu cuevavirus,LLOV)以及新型丝状病毒DH04株的血清抗体间接ELISA方法,首先构建以上五种病毒核蛋白(NP)羧基端的原核表达质粒,转化大肠杆菌BL21(DE3)进行原核表达,结果MARV、REBOV、ZEBOV和DH04四种病毒NP蛋白表达成功,鉴定为可溶性蛋白,并可与Anti-His单抗进行特异性反应,说明蛋白表达正确。然后,分别以纯化的目的蛋白为抗原包被96孔ELISA板,建立了检测四种病毒血清抗体的间接ELISA方法:优化抗原包被量为2μg/ml、封闭液为5%脱脂奶粉、二抗稀释度为1:20000。建立的ELISA方法与一种蝙蝠轮状病毒、三种蝙蝠和人的布尼亚病毒(来宾病毒、汉城病毒、春山病毒)蝙蝠感染血清均无交叉反应,证明特异性良好;并能检测1∶1 600稀释的阳性血清,说明敏感性良好;试验的批内及批间变异系数均小于10%,重复性良好。用建立的间接ELISA方法检测了云南、广西以及浙江3个地区的蝙蝠血清,总体阳性率为45.2%(141/312),其中云南阳性率明显高于其它两个省份(P0.05),浙江与广西的阳性率无明显的统计学差异。云南阳性率为65.7%(94/143),其中与DH04、REBOV、ZEBOV和MARV反应的血清分别占46.9%、36.4%、29.4%和21.0%;广西阳性率26.2%(32/122),与REBOV反应的血清最多,占16.4%,其次是DH04和ZEBOV,分别占10.7%和9.8%,MARV最少,仅占4.1%;浙江阳性率为31.9%(15/47),有27.7%的血清针对DH04反应,6.4%的血清针对ZEBOV反应,4.3%的血清针对MARV反应,2.1%的血清针对REBOV反应。根据ELISA检测结果,挑选75份蝙蝠血清,其中59份阳性血清,16份阴性血清,进行Western blot(WB)检测,发现ELISA检测为阳性的蝙蝠血清与WB检测的符合度在80%-100%之间,ELISA检测为阴性的血清与WB检测的符合度在78%-94%之间。使用单抗进行交叉反应确定,证明DH04不与REBOV、ZEBOV、MARV的抗体反应,MARV与REBOV、ZEBOV之间有较弱的交叉反应,REBOV和ZEBOV之间有很强的交叉反应。本研究成功表达了DH04、MARV、REBOV和ZEBOV的NP羧基端蛋白,在此基础上建立了检测四种丝状病毒感染血清抗体的间接ELISA方法,并对我国南方蝙蝠血清进行了丝状病毒的血清流行病学调查,结果表明我国蝙蝠存在比较广泛的丝状病毒感染,而且可能存在多种丝状病毒混合感染的情况,同时不同病毒在不同地域和蝙蝠种类上具有一定的特征:在云南和浙江DH04流行最广,而在广西REBOV流行最广;棕果蝠阳性率显著高于其它四种蝙蝠,马铁菊头蝠中针对DH04反应的血清最多,小黄蝠中针对REBOV反应的血清最多,而在南长翼蝠中未检测到阳性血清。本研究提示我国面临蝙蝠将自身携带的丝状病毒传播给其它动物的潜在风险,为了防范蝙蝠源丝状病毒病未来需要加大对蝙蝠丝状病毒病原的监测和防范措施的研究力度。
[Abstract]:Filamentous virus (Filovirus) belongs to the single stranded RNA (Mononegavirales), virales currently includes three genera, respectively (Ebolavirus), the Ebola virus belongs to the genus of Marburg virus (Marburgvirus) and Curval virus (Cuevavirus), the first two genera are mainly distributed in Africa, after a genus distributed in Europe. Filamentous virus, is highly contagious and fatal to people, so the family members of international admiral as potential biological agents, such as the Zaire Ebola virus (Zaire Ebola, virus, ZEBOV) and Marburg virus (Marburgvirus, MARV). In addition to Reston Ebola virus (Reston Ebolavirus REBOV) is the natural host of pigs and the rest of the known Ebola virus, Marburg virus and Curval virus natural host bats. Our laboratory in 2015 for the first time in Asia (Yunnan, China) discovered a new type of bat filamentous virus, that of Asia. The bats are also distributed filamentous viruses, but the species, infection rate and public health significance is not clear, and our country and there are a large number of bat species, and to establish a rapid, efficient and accurate detection method of bat filoirus infections serum and to carry out epidemiological investigation has very important scientific significance. The purpose of this study is to establish the detection of MARV, REBOV, ZEBOV, Curval (Lloviu cuevavirus, LLOV) virus, a novel filamentous virus strain DH04 serum antibody by indirect ELISA method, firstly, the above five kinds of virus nucleoprotein (NP) prokaryotic expression plasmid C-terminal, transformed into Escherichia coli BL21 (DE3) for prokaryotic expression results MARV, REBOV, four kinds of virus NP protein ZEBOV and DH04 expression, identified as soluble protein, and specific reaction with Anti-His monoclonal antibody, indicating correct expression of the protein. Then, with the purpose of purification Protein antigen ELISA 96 hole plate, an indirect ELISA was developed to detect four kinds of virus antibody: optimization of antigen amount is 2 g/ml, the closed solution of 5% skim milk, two anti dilution ELISA method established by 1:20000. and a bat rotavirus, three species of bats and people bunyavirus (guest virus, Seoul virus, spring Hill virus) infection of bat serum had no cross reaction demonstrated good specificity; and detection of 1: 1600 diluted serum showed good sensitivity; the intra batch and inter batch variation coefficient test were less than 10%, good repeatability. The developed indirect ELISA method the detection of Yunnan, the 3 areas of Guangxi and Zhejiang bat serum, the total positive rate was 45.2% (141/312), the positive rate of Yunnan was higher than that of the other two provinces (P0.05), no significant statistical positive rate in Zhejiang and Guangxi, Yunnan difference. The positive rate was 65 .7% (94/143), with DH04, REBOV, ZEBOV and MARV in serum were 46.9%, 36.4%, 29.4% and 21% in Guangxi; the positive rate was 26.2% (32/122), serum reacted with REBOV the most, accounting for 16.4%, followed by DH04 and ZEBOV, respectively accounted for 10.7% and 9.8% MARV, at least, accounted for only 4.1%; in Zhejiang, the positive rate was 31.9% (15/47), serum 27.7% response to DH04 6.4%, the serum response to ZEBOV, 4.3% to 2.1% of the serum MARV reaction, serum response to REBOV. According to the results of ELISA, selected 75 bat serum, 59 positive sera and 16 negative serum samples (blot, Western WB) detection, ELISA detected for serum and WB were detected with the bat between 80%-100%, ELISA and WB for the detection of serum test negative coincidence rate between 78%-94%. Using monoclonal antibody cross reactivity to determine, proof of DH04 and REBOV, ZEBOV, MARV and MARV antibody response, RE BOV, a weak cross reaction between ZEBOV, a strong cross reaction between REBOV and ZEBOV. The expression of DH04, MARV, NP and REBOV protein carboxyl terminal ZEBOV, established on the basis of indirect ELISA for serum antibody detection of four species of filamentous virus infection, and the South China bat serum a seroepidemiological survey of filamentous viruses, the results show that China's bat more extensive filamentous virus infection, but there may be a variety of filamentous virus mixed infection, while different virus has certain characteristics in different regions and bat species: Zhejiang DH04 in Yunnan and the most popular, the most popular REBOV in Guangxi; the positive rate of Rousettus was significantly higher than that of other four species of bats, Rhinolophus ferrumequinum in serum response to DH04 most, in response to REBOV yellow bat serum most, while in the South long winged bats in unghecded The positive serum was detected. This study indicates that our country is facing the potential risk that bats transmit their filamentous virus to other animals. In order to prevent bat's filamentous disease, we need to intensify the research on monitoring and prevention measures of bat's filamentous virus.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.5

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