蝙蝠携带狂犬病毒检测方法的建立及在中国南方的调查研究

发布时间：2018-05-06 08:01

本文选题：蝙蝠 + 狂犬病毒　；参考：《南方医科大学》2009年博士论文

【摘要】：1.研究背景和目的: 狂犬病(Rabies)是人畜共患的自然疫源性疾病,可引起人和所有温血动物急性致死性脑脊髓炎,一旦发病,患者或病畜几乎100%死亡。狂犬病可在世界范围内分布,是全球性的严重公共卫生问题。狂犬病毒(Rabies virus)是人和动物狂犬病的病原,已知动物宿主众多,且随着研究的深入,越来越多的物种都能分离出狂犬病毒。自1953年美国首次发现食虫蝙蝠咬伤引起的人狂犬病以来,蝙蝠携带狂犬病毒及其与人狂犬病的关系引起人类的广泛关注,近年来许多国家和地区不断地从蝙蝠体内分离出狂犬病毒。狂犬病的主要传染源是野生动物,主要包括:狼、狐、豹、野狗、猴、猫、臭鼬、浣熊和蝙蝠等。狂犬病的流行一般先出现在野生动物中间,通过家养动物波及人类。家养动物是野生动物狂犬病和人类狂犬病的联系环节。犬虽然非最敏感动物,但是由于世界大多数地区的城市和农村养犬密度很高,所以犬已成为病毒的长期宿主和媒介。家猫亦有同样的趋势。狂犬病的传染源在不同地区有所不同,如非洲85%以上的国家、南美大多数国家和亚洲地区国家(除以色列和孟加拉以外)以犬狂犬病为主,而南非、欧洲、美国和加拿大等北美地区则主要是野生动物狂犬病。圭亚那、巴拿马和多哥则是以蝙蝠传播狂犬病。荷兰、德国和西班牙的蝙蝠狂犬病仅次于野生动物狂犬病。我国绝大多数的狂犬病为犬所致,偶有猫、牛等其他动物的报道。近年来,野生动物狂犬病的比重不断增加,时有野生动物狂犬病传给人的报道出现,野生动物携带狂犬病毒带来的危害日趋严重。野生动物不仅直接传播疾病,而且可能通过破坏畜牧业间接地影响人类健康,而野生动物的管理比较困难,一般地难于对其进行免疫预防。尤其是某些蹄类野生动物具有迁徙的本能,能周期性地、替换方向地改变栖息地以获得最佳的环境条件。鉴于这个特点,野生动物作为狂犬病毒的贮存宿主和媒介,能给传播狂犬病毒带来更大地危害。蝙蝠属翼手目(Chiroptera)动物,是哺乳类中分布最广、数量最多的动物之一,全世界大约有17科180属,约960种,其中我国有7科29属107种。翼手目分两个亚目:大蝙蝠亚目(Megachiroptera),又称狐蝠,我国有1科5属7种;小蝙蝠亚目(Microchiroptera),即通常所说的蝙蝠,我国有6科24属100种。除南北极外,蝙蝠在世界各地都有分布,包括在高纬度地区、荒凉的沙漠和孤立的岛屿上,甚至在撒哈拉大沙漠也有蝙蝠的活动。然而,大多数蝙蝠种类生活在热带和亚热带地区。蝙蝠物种丰富,分布广泛,寿命长,并有很强的飞行能力,其中一些蝙蝠种类还有长途迁移的习性。蝙蝠与人类接触密切。有些蝙蝠就栖息在屋檐下,或是房屋的裂缝中;另外蝙蝠在觅食时经常会误闯入居民家中;一些旅游景点的岩洞中也居住有蝙蝠。在中医里面,菊头蝠的粪便被做成“夜明沙”用于治疗眼疾;在广东,果蝠被一些餐馆作为野味摆上餐桌;湖南一些地区则相信蝙蝠可以用来治疗癫痫。狂犬病病毒(Rabies virus)为弹状病毒科(Rhabdoviridae)狂犬病毒属(Lyssavirus),形态呈弹状(60～400 nm×60～85 nm),一端纯园,一端平凹,有囊膜,内含衣壳呈螺旋对称。核酸是单股负链不分节段RNA,基因组长约12 kb,从3′到5′端依次为N、P、M、G、L的5个基因,各个基因间还含非编码的间隔序列。分别编码核蛋白(N)、磷蛋白(P)、基质蛋白(M)、糖蛋白(G)、转录酶大蛋白(L)。完整的病毒颗粒由核衣壳及包膜两部分构成,核衣壳由RNA与核蛋白、磷蛋白和转录酶大蛋白构成,而包膜由糖蛋白和基质蛋白构成。在狂犬病毒的5个基因中,N基因有高度保守性。1993年Bourhy等根据核蛋白基因N端500个碱基的同源性将狂犬病毒属分为6个基因型,基因1～4型分别对应于血清Ⅰ～Ⅳ,而血清Ⅴ型的欧洲狂犬病毒EBL1株和EBL2株为基因型5、6。1998年澳大利亚报道分离出新的蝙蝠狂犬病病毒,被定为基因7型,澳大利亚蝙蝠狂犬病病毒(ABLV)。近年来,新基因型别的狂犬病病毒(Lyssavirus)不断发现,在蝙蝠身上分离到Aravan virus,Khujand virus,Irkutsk virus,以及WestCaucassian bat virus(WCBV)几种狂犬相关病毒。基因1型病毒为狂犬病毒,其他基因型病毒称为狂犬病相关病毒。这些基因型又进一步合并为2个遗传谱系(phylogroup):基因型1、4、5、6和7为遗传谱系Ⅰ;基因型2和3为遗传谱系Ⅱ。蝙蝠是狂犬病较独特的宿主和媒介,除基因3型狂犬相关病毒外狂犬病毒属各基因型均可在蝙蝠体内分离得到。我国是受狂犬病危害最为严重的国家之一,我国华南地区及部分华中地区近几年狂犬病的发病数呈快速上升趋势。这些地区蝙蝠分布数量较大,种类多,且蝙蝠夏天活动冬天冬眠的特点与狂犬病春夏秋发病多季节特点有关。这些现象提示我国蝙蝠可能作为狂犬病毒的宿主。然而,目前我国这一方面的研究还比较少。我国蝙蝠携带狂犬病毒的情况尚不清楚,因此有必要开展研究。 2研究方法 2.1建立狂犬病毒免疫学和分子生物学检测方法 2.1.1直接免疫荧光技术(The direct fluorescent antibody test,DFA)检测狂犬病毒 ①用狂犬病毒减毒活疫苗颅内接种小鼠,选择抗体工作浓度。 ②用选择的抗体浓度对蝙蝠脑组织进行检测。 2.1.2巢式RT-PCR检测狂犬病毒 ①参照论文,以特异性扩增核蛋白(N)基因最保守区域为目的基因,比对了7个基因型的狂犬病毒,设计了套式RT-PCR检测引物。 ②用狂犬病毒减毒活疫苗作为阳性对照,进行巢式RT-PCR最低检测浓度试验。 ③巢式RT-PCR对蝙蝠脑组织狂犬病毒的检测。 2.1.3初步建立RT-LAMP(RT-loop-mediated isothermal amplification)快速检测方法检测狂犬病毒 ①参照论文,以特异性扩增核蛋白(N)基因最保守区域为目的基因,比对了7个基因型的狂犬病毒,自行设计RT-LAMP检测引物。 ②用狂犬病毒减毒活疫苗作为阳性对照,进行RT-LAMP最低检测浓度试验。 2.2蝙蝠标本的采集 2007年5月至2007年8月之间,2008年7月至2008年8月之间,在海南、广东和湖南部分地区共收集蝙蝠1057只,进行编号后,请广州大学生命科学院蝙蝠研究专家吴毅教授进行鉴定。采集蝙蝠的地方主要有废弃的房屋,或者房屋的缝隙、屋檐,以及野外阴湿的山洞。通过无菌操作解剖蝙蝠,取完整脑组织标本,一份标本分为三种保存方式。分别保存在含有RNAlater、PBS及含10%甲醛的PBS缓冲盐溶液的冻存管中,4℃运输,回实验室后,放入-70℃保存。 2.3蝙蝠脑组织样本狂犬病毒的检测用上述建立的DFA和巢式RT-PCR方法对采集的蝙蝠脑组织进行检测。 2.4测序和基因分析 ①对于扩增获得的目的基因进行测序; ②测序结果利用GeneBank的BLAST软件进行在线同源性分析; ③在GeneBank下载其他狂犬病毒序列,利用Clustal W进行多序列比对分析; ④用MEGA 4.0软件,采用Neighbor-Joining法,Jukes-Cantor模型做进化树分析。 2.5病毒分离培养检出阳性样本用Vero细胞进行病毒分离培养,结果用流式细胞术和细胞免疫荧光方法进行鉴定。 2.6质量控制 ①移液枪头、EP管、冻存管、手套等实验耗材均为一次性用品; ②逆转录过程采用的移液枪头、EP管和研磨器用DEPC处理,高温高压消毒,烤干后待用,以去除RNA酶污染; ③RNA的提取,RT-PCR的反应体系的试剂加样均在无菌间生物安全Ⅱ级超净台进行操作,防止环境中RNA酶的污染; ④操作过程中,全程戴口罩、帽子,勤换手套,防止样本和试剂受到来自操作者携带的RNA酶污染; ⑤在RNA提取,RT-PCR,直接免疫荧光过程中均设立阳性对照和阴性对照。 3结果 3.1狂犬病毒直接免疫荧光检测方法的建立:直接免疫荧光法检测狂犬病毒,筛选免疫荧光抗体工作浓度为1:1×10~2,该浓度下所有阳性对照均能检出,而阴性对照未检出。可在蝙蝠脑组织中检出狂犬病毒。 3.2巢式RT-PCR检测方法的建立:应用巢式RT-PCR方法对狂犬病毒进行检测,能检测到狂犬病毒减毒活疫苗(20.0 LD_(50)/0.03 ml)最低浓度为1:1×10~4。空白对照及阴性对照均未检出,灵敏度、特异度均较高。可检出蝙蝠脑组织中的狂犬病毒。 3.3 RT-LAMP检测的初步建立:应用RT-LAMP方法对狂犬病毒进行检测,能检测到狂犬病毒减毒活疫苗(10.0 LD_(50)/0.03 ml)最低浓度为1:1×10~4。空白对照及阴性对照均未检出,灵敏度、特异度均较高。初步建立狂犬病毒的快速检测方法,用于蝙蝠脑组织检测,与巢式RT-PCR相比有较高的灵敏度,且大大地缩短了检测时间。 3.4病毒分离培养后鉴定:2007年采集的30例狂犬阳性样本悬液转染Vero细胞7 d后,作流式细胞术检测,加入荧光标记的狂犬病毒单克隆抗体后,平均荧光强度增强5.34～27.22,95%可信区间10.62～14.66;荧光细胞结合百分率增加3.48%～11.74%,95%可信区间5.52%～7.36%。2008年采集的9例阳性脑组织样本制成悬液后,转染细胞用荧光标记抗体结合后,在显微镜下可见苹果绿荧光,而未加入荧光抗体细胞空白对照未见荧光。 3.5现场调查应用:应用直接免疫荧光和巢RT-PCR两种检测方法对采集的蝙蝠脑组织标本进行检测,在普通长翼蝠、中菊头蝠和小黄蝠中都检测出了狂犬病毒。巢式RT-PCR法检测阳性率为3.69%(39/1057),其中第一年阳性率为6.41%(30/468),第二年阳性率为1.53%(9/589),检出自湖南,均从普通长翼蝠和中菊头蝠中检出。对2008年采集的589份蝙蝠脑组织样本,直接免疫荧光方法检出率为9.85%(58/589)。阳性样本检出自湖南的普通长翼蝠、中菊头蝠和海南的小黄蝠。 3.6对序列进一步的系统进化分析表明:尽管样本间存在基因多态性,但是构建的系统进化树显示本次所检测的蝙蝠狂犬病毒聚集成一个分支,都属于狂犬病毒基因Ⅰ型。 4结论 4.1蝙蝠脑组织携带狂犬病检测方法的建立:建立了直接免疫荧光和巢式RT-PCR两种方法实验室检测狂犬病毒,建立的方法可以用于蝙蝠脑组织狂犬病毒的检测。两种方法在检测狂犬病毒上均有较高的特异度;初步比较直接免疫荧光方法和巢式RT-PCR方法检测蝙蝠脑组织携带狂犬病毒灵敏度有所不同,直接免疫荧光较高。初步建立的RT-LAMP方法能够快速检测蝙蝠脑组织狂犬病毒。 4.2用所建立的两种方法对中国南方三省的蝙蝠进行现场流行病学调查应用,检出湖南中菊头蝠、普通长翼蝠和海南小黄蝠三种体内存在狂犬病毒。但不能排除其他种类蝙蝠携带狂犬病毒的可能。 4.3本次研究检测出的狂犬病毒序列差异不大,全部包括在CHINA-1分支,属于基因1型狂犬病毒。与亚洲流行的基因型别一致。
[Abstract]:1. background and purpose of the study:
Rabies (Rabies) is a natural epidemic disease of zoonotic disease, which can cause acute fatal encephalomyelitis in human and all warm blooded animals. Once the disease occurs, the patient or the sick animal is almost 100%. The rabies can be distributed worldwide and is a global serious public health problem. The Rabies virus is the pathogen of human and animal rabies. There are many known animal hosts, and more and more species are able to separate rabies virus with the study. Since the first discovery of the human rabies caused by the bite of insectivorous bats in the United States in 1953, the bats carrying rabies virus and its relationship with human rabies have aroused widespread concern in human beings. In recent years, many countries and regions have continuously been from many countries and regions. The bats are isolated from the rabies virus.
The main source of rabies infection is wild animals, including wolves, foxes, leopards, wild dogs, monkeys, cats, skunk, raccoons and bats. The epidemic of rabies is usually found in the middle of wild animals and through domesticated animals to humans. Domesticated animals are the link between wild animal rabies and human rabies. Although dogs are not the most sensitive animals, but not the most sensitive animals, but Due to the high density of dogs in urban and rural areas in most parts of the world, dogs have become the long-term host and vector of viruses.
The source of rabies is different in different regions, such as more than 85% of Africa, most countries in South America and Asian countries (except Israel and Bengal) are mainly canine rabies, while South Africa, Europe, the United States and Canada are mainly wild rabies. Guyana, Panama and Togo are the most important ones. Bats spread rabies. Holland, Germany and Spain are second only to wild animal rabies. Most of the rabies in our country are caused by dogs. There are occasional reports of other animals, such as cats and cattle.
In recent years, the proportion of wild animal rabies has been increasing, and the reports of wild animal rabies have been reported to people, and the harm of wild animals carrying rabies virus is becoming more serious. Wild animals not only direct the disease directly, but also may indirectly affect the human health by destroying animal husbandry, and the management of wild animals is difficult, In general, it is difficult to immunize them. Especially, some hoofed wild animals have a migratory instinct, which can periodically change their habitats in a replacement direction to obtain the best environmental conditions. In view of this, wildlife as the storage host and medium of the rabies virus can cause more harm to the spread rabies virus.
The Chiroptera animal is one of the most widely distributed and most abundant mammals in mammals. There are about 17 families and 180 genera in the world, about 960 species, of which there are 7 families, 29 genera and 107 species in our country. The bats are divided into two suborders: the large bat suborder (Megachiroptera) and the fox bat, our country has 1 families, 5 genera and 7 species; the small bat suborder (Microchiroptera) is usually the common species. There are 6 families, 24 genera and 100 species of bats.
In addition to the north and south poles, bats are distributed all over the world, including in high latitudes, desolate deserts and isolated islands, and even in the Sahara desert. However, most bats live in tropical and subtropical areas. Bats are rich in species, widely distributed, long life, and have strong flying abilities. Some bats have a long migration habit.
Bats live in close contact with humans. Some bats perch under eaves, or cracks in houses; and bats often break into homes in foraging; bats are also inhabited in caves in some tourist attractions. In traditional Chinese medicine, the dung of chrysanthemum bat is made into "night clear sand" for treatment of eye diseases; in Guangdong, fruit bats are in restaurants. In Hunan, some areas believe that bats can be used to treat epilepsy.
Rabies virus is a rabies virus (Rhabdoviridae) rabies virus (Rhabdoviridae) rabies virus (Lyssavirus), with a projectile shape (60~400 nm x 60~85 nm), one end pure garden, a flat end, a capsule, and a spiral symmetry in the capsid. The nucleic acid is a single strand of negative segment RNA, and the gene group is about 12 KB, and the 3 'to 5' ends are N, P, M, G, 5 bases. Because each gene contains an uncoded interval sequence, encoding nucleoprotein (N), protein (P), matrix protein (M), glycoprotein (G), and transcriptional large protein (L). The complete virus particles are composed of two parts of the nucleocapsid and envelope, and the nucleocapsid is composed of RNA and nuclear egg white, protein and transcriptional protein, and the envelope is composed of glycoprotein and matrix protein. Of the 5 genes of rabies virus, the N gene has a highly conserved.1993 year Bourhy and so on, according to the homology of the 500 bases of the nucleoprotein gene, the rabies virus is divided into 6 genotypes, and the gene 1~4 corresponds to the serum I to IV respectively, while the serotype V type European rabies virus EBL1 and EBL2 strain is the genotype 5,6.1998 Australian Australian Dali. Subreports isolated the new bat rabies virus (ABLV), the Australian bat rabies virus (ABLV). In recent years, the new gene type of rabies virus (Lyssavirus) has been found to separate Aravan virus, Khujand virus, Irkutsk virus, and WestCaucassian bat virus (WCBV) virus related viruses in the bats. The gene type 1 virus is a rabies virus, and the other genotypes are called rabies related viruses. These genotypes are further merged into 2 genetic lineages (phylogroup): genotype 1,4,5,6 and 7 are genetic lineage I; genotype 2 and 3 are genetic lineage II. Bats are the more specific hosts and vectors of rabies, except for the virus type 3 rabies related viruses. All rabies virus genotypes can be isolated from bat.
China is one of the countries most seriously affected by rabies. The number of rabies in Southern China and central China has been rising rapidly in recent years. These areas have a large number of bats and many species, and the characteristics of the winter hibernation in the summer activities of bats are related to the seasonal characteristics of rabies in spring, summer and autumn. Bats in our country may be used as the host of rabies virus. However, there are few studies on this aspect in our country at present. It is not clear that bats are carrying rabies virus in our country, so it is necessary to carry out the research.
2 research methods
2.1 to establish rabies virus immunology and molecular biology detection methods.
Detection of rabies virus by 2.1.1 The direct fluorescent antibody test (DFA)
First, the live attenuated rabies virus vaccine was injected into the mice and the antibody concentration was selected.
(2) using the selected antibody concentration to detect the brain tissue of bat.
Detection of rabies virus by 2.1.2 nested RT-PCR
(1) according to the paper, using the most conservative region of the specific amplified nucleoprotein (N) gene as the target gene, a set of primers for RT-PCR detection were designed to compare the 7 genotypes of rabies virus.
(2) using the live attenuated rabies vaccine as the positive control, the nested RT-PCR minimum concentration test was conducted.
Nested RT-PCR for detection of rabies virus in bat brain tissue.
2.1.3 preliminary establishment of RT-LAMP (RT-loop-mediated isothermal amplification) rapid detection method for rabies virus detection
(1) according to the paper, the most conservative region of the specific amplified nucleoprotein (N) gene was used as the target gene, and the 7 genotypes of rabies virus were compared, and RT-LAMP primers were designed by ourselves.
(2) using the live attenuated rabies vaccine as the positive control, the minimum RT-LAMP concentration test was carried out.
Collection of 2.2 bats
From May 2007 to August 2007, between July 2008 and August 2008, 1057 bats were collected in parts of Hainan, Guangdong and Hunan. After numbering, Professor Wu Yi, a bat research expert at the Guangzhou University Life Science Academy, was asked to identify the places where the bats were mainly abandoned, or the gaps, eaves, and wilderness of the houses. The whole brain tissue specimens were dissected by aseptic operation. A specimen was divided into three preservation methods. They were stored in the cryopreservation tube containing RNAlater, PBS and 10% formaldehyde containing PBS buffer solution, and were transported to the laboratory at 4, and stored at -70 C after returning to the laboratory.
Detection of rabies virus in 2.3 bats brain tissue samples
The DFA and nested RT-PCR methods were used to detect the brain tissues of the bat.
2.4 sequencing and gene analysis
(1) sequencing of the target genes obtained by amplification;
(2) sequencing results were analyzed by online homology analysis using GeneBank's BLAST software.
(3) downloading other rabies virus sequences in GeneBank, and using Clustal W for multiple sequence alignment analysis.
(4) using MEGA 4 software, Neighbor-Joining method and Jukes-Cantor model were used to analyze phylogenetic tree.
2.5 virus isolation and culture
The positive samples were detected by Vero cells. The results were identified by flow cytometry and cellular immunofluorescence.
2.6 quality control
(1) all the experimental consumables such as pipette head, EP tube, freezing tube and gloves are disposable goods.
(2) the pipette head used in the reverse transcription process, the EP tube and the grinder were treated with DEPC, then sterilized at high temperature and high pressure, dried and then used to remove RNA enzyme pollution.
(3) the extraction of RNA and the reagent addition of RT-PCR reaction system are operated at the aseptic biosafety stage II super clean platform to prevent the pollution of RNA enzymes in the environment.
During the operation, wearing masks, hats and gloves should be worn throughout the operation to prevent samples and reagents from being contaminated by RNA enzymes from operators.
5. Positive control and negative control were established in RNA extraction, RT-PCR and direct immunofluorescence.
3 Results
The establishment of 3.1 rabies virus direct immunofluorescence detection methods: direct immunofluorescence detection of rabies virus, screening immunofluorescence antibody working concentration of 1:1 x 10~2, the concentration of all positive controls can be detected, but the negative control is not detected.
3.2 the establishment of nested RT-PCR detection method: the nested RT-PCR method was used to detect rabies virus, and the lowest concentration of rabies virus attenuated vaccine (20 LD_ (50) /0.03 ml) was detected, and the lowest concentration was 1:1 x 10~4. blank control and negative control, and the sensitivity and specificity were all high.
The preliminary establishment of 3.3 RT-LAMP detection: using RT-LAMP method to detect rabies virus, the lowest concentration of rabies virus attenuated vaccine (10 LD_ (50) /0.03 ml) was detected, the minimum concentration was 1:1 x 10~4. blank control and negative control, and the sensitivity and specificity were all high. Weaving detection has higher sensitivity compared with nested RT-PCR, and greatly reduces detection time.
3.4 virus isolation and culture identification: 30 cases of rabies positive sample suspension collected in 2007 were transfected to Vero cells for 7 d and were detected by flow cytometry. The average fluorescence intensity increased from 5.34 to 27.22,95% confidence interval 10.62 to 14.66, and the fluorescein binding percentage increased 3.48% to 11.74%, 95% trusted. After a suspension of 9 positive brain tissue samples collected in the interval from 5.52% to 7.36%.2008, the transfected cells were combined with fluorescent labeled antibodies, and the apple green fluorescence was observed under the microscope, but no fluorescence was found in the blank control of the fluorescent antibody cells.
3.5 field investigation application: using direct immunofluorescence and nest RT-PCR two detection methods to detect the collected bats' brain tissue specimens, the rabies virus was detected in ordinary long wing bats, middle chrysanthemum bats and small yellow bats. The positive rate of nested RT-PCR method was 3.69% (39/1057), and the positive rate of the first year was 6.41% (30/468) and second year positive. The sex rate was 1.53% (9/589), detected from Hunan, all from the common long wing bats and the middle chrysanthemum bats. The direct immunofluorescence detection rate of 589 bats collected in 2008 was 9.85% (58/589). The positive samples were detected from the common long wing bats, the middle chrysanthemum bats and the Yellow bats in Hainan.
3.6 further phylogenetic analysis of sequences showed that although polymorphisms existed among samples

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R181.3

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