湘琼两地蝙蝠携带星状病毒与诺如病毒的调查研究

发布时间：2017-12-31 20:13

本文关键词：湘琼两地蝙蝠携带星状病毒与诺如病毒的调查研究　出处：《南方医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：研究背景和目的研究表明,蝙蝠是多种人兽共患疾病病毒的自然宿主,并可以跨越物种间屏障,将病毒传播给人类和其他动物。目前研究人员已经从蝙蝠身上发现或分离很多种病毒,约有18个病毒科包含70多种病毒。已知能从蝙蝠传播给人类的病毒主要有狂犬病毒、尼巴病毒、亨德拉病毒,还有类SARS冠状病毒,这些病毒都可以引起人类致命性的疾病。其他病毒,如甲病毒属、黄病毒属和布尼亚病毒属病毒可通过节肢动物传播给蝙蝠,但还不确定蝙蝠是不是这类病毒所致疾病的播散宿主。可见,还需要大量研究,以明确蝙蝠在病毒性自然疫源性疾病中所扮演的角色。我国约有120种蝙蝠,但国内对蝙蝠的研究工作起步较晚,对其生态学、行为学研究尚不够深入,尤其是对我国蝙蝠携带哪些种类病毒,以及病毒与蝙蝠生态学的关系,对人类健康的影响的研究还较少。迄今为止,我国科研工作者对蝙蝠所携带病毒的研究有以下发现：从蝙蝠体内分离到罗斯河病毒、乙型脑炎病毒、基孔肯雅病毒、森林脑炎病毒、SARS类冠状病毒、狂犬病病毒、星状病毒、尼巴病毒等。这些发现表明我国蝙蝠可携带某些我国常见传染病的病毒。病毒性胃肠炎(viral gastroenteritis)又称病毒性腹泻,是一组由多种病毒引起的急性肠道传染病。临床特点为起病急、恶心、呕吐、腹痛、腹泻,排水样便或稀便,也可有发热及全身不适等症状,病程短,病死率低。病毒性腹泻在儿童中更是一种常见病和多发病,是导致儿童死亡的重要原因。引起儿童腹泻的病毒主要有轮状病毒(rotavirus)、肠道腺病毒(adenovirus)、星状病毒(astrovirus, Astv)、诺如病毒(norovirus, NoV)等。患者与无症状带毒者是病毒性胃肠炎的主要传染源。粪-口途径传播是病毒性胃肠炎最重要的传播途径,少部分可以通过空气传播,其可通过被污染的食物或水引起人群胃肠炎暴发或散发。回顾上世纪90年代的亨德拉病毒和尼帕病毒的暴发事件,也是源于食源性感染,是由于蝙蝠病毒污染了水域、水果,再依次传播给马、猪、人,最后导致疾病暴发。星状病毒(Astrovirus, Astv)于1975年在胃肠炎患儿粪便标本中首次发现,由于电镜下病毒颗粒呈星形,因此被命名为星状病毒。此后世界各地均已有星状病毒感染的报道,它既可散发也可以引起暴发流行,亦可引起医源性感染。住院腹泻患儿中星状病毒检出率为2.5%-9.0%,目前认为星状病毒是婴幼儿病毒性胃肠炎的第二位病因,仅次于轮状病毒。星状病毒既可感染人,也可感染牛、羊、猪、狗、鹿、火鸡、鸭、鼠等多种动物,导致不同程度的胃肠炎。人群感染的星状病毒,传统上可分为8个血清型。最近,研究者又在腹泻病人中发现两种的新星状病毒,astrovirus MLB和astrovirus VA。研究还发现,Astv MLB 1与新近发现的鼠星状病毒很相近;Astv VA1在进化树上与感染羊和貂的星状病毒最为接近。这提示新发现的人星状病毒可能和动物的星状病毒存在一定的关系。近年来,香港大学研究小组分别从香港和中国大陆的蝙蝠体内检出星状病毒,检出率高达46%和44.8%；基因分析显示蝙蝠星状病毒具有高度基因多态性,部分病毒可能与感染人的星状病毒有系统进化的关系。然而关于蝙蝠星状病毒的生态学资料还很稀少,有必要进一步扩充不同地区和不同种类的蝙蝠样本进行研究。诺如病毒(Norovirus, NoV)是一组杯状病毒属病毒,其原型株诺瓦克病毒于1972年在美国诺瓦克市被分离发现。由于该组病毒极易变异,此后在其他地区又相继发现并命名了多种类似病毒,统称为诺如病毒。NoV是一种高致病的、传染性极强的肠胃病毒,能导致急性胃肠炎,一旦有人感染,通常会发展为群体性的大规模传染。遗憾的是,由于病毒的高度变异性及其缺少合适的动物研究模型,现今还未研发出理想的NoV疫苗,患者容易发生反复感染。自然界中携带NoV的物种很多,包括有人、猪、牛、鼠、海狮等,可导致它们发生不同程度的症状。根据基因特征,NoV被分为五个遗传群(Gene Group, GG):GGⅠ、GGⅡ、GGⅢ、GGⅣ和GGV。五个遗传群中,GGⅠ、GGⅡ和GGIV主要感染人类,GGⅢ感染牛,GGV则在鼠类动物内检出。虽然研究已表明NoV在多种动物体内广泛存在,但是关于动物NoV感染的流行病学还不太清楚。动物NoV在动物体内感染率差别较大,研究发现北美的实验鼠鼠类NoV的感染率高达22.1%,英国检测发现牛的腹泻病例中有11%比例为NoV感染所致,而猪NoV检出率比较低,在日本猪GGⅡNoV的检测率仅为0.35%,在荷兰检测率为2.0%。尽管现今没有证据表明动物NoV可以感染人类,但是分子生物学分析表明动物NoV和人类NoV的毒株相互之间有密切的关系,尤其是猪NoV的毒株可被归入到与人类NoV毒株相同的基因群(GGⅡ)。新近研究发现猪体内普遍存在人NoV的抗体,这进一步加强了动物可能充当着人类NoV储存库的假设。此外,研究还发现贝壳类动物可同时携带人类NoV和动物NoV,这提示一种新的基因重组株可能出现。最近,在加拿大人们检测到猪NoV和牛NoV的序列与人NoVGGⅡ.4毒株的序列非常接近。因此,将来或许要重新评估NoV种间传播的可能性及其人畜共患的风险。 2009年,武汉病毒研究所在蝙蝠体内检测到另一种导致人腹泻的病毒—肠道腺病毒,检出率为8%(28/350),基因分析显示蝙蝠腺病毒亦具有高度基因多样性,这提示蝙蝠可能有携带一系列高变异病毒的能力。研究已经在蝙蝠体内发现了星状病毒和腺病毒两种腹泻相关病毒,关于蝙蝠是否携带有NoV,暂未见有文献报道,因而我们不禁疑问蝙蝠是否携带有可导致爆发流行的NoV。鉴于NoV的高度基因变异性和多宿主性,作为种类第二多的哺乳动物(1000多种)一蝙蝠也很有可能携带此类病毒,甚至可能是它们的宿主。因此,为进一步了解不同地区不同种类蝙蝠携带星状病毒的本底情况,探讨其与感染人星状病毒的关系,同时了解这些地区的蝙蝠是否携带诺如病毒,本研究拟在先前未见报道的湖南、海南部分地区为现场,对当地蝙蝠携带星状病毒和诺如病毒情况进行流行病学调查。研究方法 1.样本的采集和处理 2007年7月至2008年7月,于岳阳、邵阳、海口三地区蝙蝠长期监测点中选取4个蝙蝠自然栖息地,现场捕捉蝙蝠,进行编号后,请蝙蝠研究专家进行鉴定。标本采集方式分两种,分别为采集肛拭子和剪取直肠组织。采集的样本存放于含200μl RNAlater储存液的冻存管中,置放于密封的冰袋泡沫盒中当天运回实验室,放于-80℃冰箱贮存,待检。 2.蝙蝠病毒的检测与鉴定2.1星状病毒 ①半巢式PCR筛检样本参照文献,针对星状病毒高度保守的RdRp基因,设计合成5条用于巢式PCR的引物,包括4条前引物和1条共同的后引物,应用巢式PCR方法检测标本。 ②序列鉴定与分析对阳性PCR产物进行切胶、纯化和回收,然后送样至公司测序。如遇阳性PCR产物条带很弱者,则先做克隆,再取接种培养后菌液送样至公司测序鉴定。 ③将测序结果导入GenBank的BLAST软件进行在线同源性分析,然后在GenBank下载相关核酸序列,利用Clastal_X软件对所要分析的序列进行多序列比对分析,并用DNAstar软件对核酸序列相似性进行计算。使用MEGA4.1软件,用Neighbor-Joining法构建进化树进行分析。 2.2诺如病毒 ①TaqMan real-time PCR检测基因组Ⅱ型诺如病毒(GGⅡNoV) 参照文献并合成用于检测GGⅡNoV公认的特异引物和探针,利用TaqMan real-time PCR方法对本进行检测。 ②SYBR Green real-time PCR检测诺如病毒参照文献合成可检测NoV的两对通用引物P289/P290和12Y/13I,应用SYBR Green real-time PCR方法分别检测标本。 ③GGV型NoV特异引物检测标本参照文献合成公认的可检测GGV型NoV引物,应用普通RT-PCR方法检测标本。 ④对普通RT-PCR扩增获得的可疑阳性PCR产物进行切胶、纯化和回收,然后送样至公司测序。对TaqMan real-time PCR检测的可疑阳性PCR产物,送样至公司克隆鉴定。 3.质量控制 ①移液枪头、冻存管、EP管等实验耗材均为一次性用品。 ②RNA提取和逆转录实验所采用的所有容器和配制试剂用DEPC水处理,高温高压处理,烘干待用,以去除RNA酶污染。 ③全程戴口罩、手套、帽子,在无菌室生物安全柜进行操作,防止试剂和实验过程受到污染。 ④实验过程严格设立阳性对照、阴性对照和空白对照。结果 1蝙蝠基本情况本次研究在湖南、海南两省四个监测点共采集蝙蝠321只,涉及2个蝙蝠亚目4个蝙蝠科9个种类的蝙蝠。共制备蝙蝠粪便标本321份,包括肛拭子59份和直肠样本262份。 2星状病毒的检测 ①巢式RT-PCR检测到阳性标本29例,总阳性率为9.03%(29/321),在9种表面健康的蝙蝠中发现有3种蝙蝠携带有星状病毒,阳性蝙蝠种类包括长翼蝠(22/187)、小黄蝠(6/38)和棕果蝠(1/59),以小黄蝠的检出率最高。 ②以星状病毒RdRp基因部分氨基酸序列构建的进化树分析显示,本研究检测的蝙蝠星状病毒不仅与来源于其他种类的星状病毒差异较大,而且本身也有很大的差异。然而,有5个蝙蝠星状病毒却与羊星状病毒(ovine Astv)、貂星状病毒(mink Astv)和人新型星状病毒Astv VA在进化树上最为相近,序列相似性为51.6%-63.1%。另外,有8株从邵阳地区同一个洞穴长翼蝠检测到的星状病毒相聚在一小分支,它们的氨基酸序列相似性为97%-99%。 3诺如病毒的检测本研究采用TaqMan real-time PCR、SYBR Green real-time PCR和普通RT-PCR三种实验方法,应用多对引物分别筛查上述蝙蝠粪便样本GGⅠ-Ⅳ型NoV携带情况,检测结果均为阴性。结论 ①巢式PCR检测到星状病毒阳性率为9.03%(29/321),并首次在果蝠身上检出星状病毒(1/59),提示此类星状病毒可能适应于翼手目动物,尽管不同种类的蝙蝠对它的易感程度不一。 ②本研究检出的蝙蝠星状病毒基因多态性丰富,这结果与先前研究一致。5株蝙蝠星状病毒却与羊星状病毒、貂星状病毒、人星状病毒Astv VA在进化树上最为相近,并具有较高的氨基酸序列相似性。这提示蝙蝠星状病毒可能跨种族传播给人类或其他动物,亦或是在它们之间相互传播。 ③来自邵阳同一个洞穴检测得到的8株星状病毒具有高度的基因相似性,进一步提示蝙蝠星状病毒在同一个洞穴内传播的可能。 ④本研究从所收集的321份蝙蝠粪便样本中未检测到诺如病毒,提示蝙蝠携带诺如病毒的可能性不大。由于诺如病毒高度遗传变异,而且现今没有一对单独引物可同时检测出全部五个基因组的诺如病毒。蝙蝠是否携带诺如病毒还不清楚,有待进一步研究。
[Abstract]:Background and purpose of research
Research shows that bats are many zoonotic virus natural host, and can cross the species barrier and spread the virus to humans and other animal. At present, researchers have found that separation from the bats or many types of viruses, about 18 virus, including more than 70 kinds of virus. Known to spread to humans from bats the virus mainly has the rabies virus, Nipah virus, Hendra virus, and SARS coronavirus, these viruses can cause human fatal disease. Other viruses, such as a virus, and the virus flavivirus bunyavirus by arthropod animal spread to bat, but it is not clear whether bats are of this kind of disease caused by virus the spread of the host. Obviously, we still need a lot of research, in order to clear the bat in viral disease of natural focus in the role.
There are about 120 species of bats in China, but the domestic research work on bat started late, the ecology, behavioral research is still not enough, especially on what kind of virus carrying our bat, the relationship between virus and bat ecology, study the impact on human health is less. So far, the research in our country scientists are carrying the virus of bats are as follows: from separation to bat in the Ross River virus, Japanese encephalitis virus, chikungunya virus, Semliki Forest virus, SARS coronavirus, rabies virus, astrovirus, Nipah virus. These findings suggest that China's bat can carry some of common infectious diseases in China virus.
Viral gastroenteritis (viral gastroenteritis) also known as viral diarrhea, is a group of acute intestinal infectious disease caused by a variety of viruses. The clinical features of acute onset, nausea, vomiting, abdominal pain, diarrhea, drain or rare, also can have fever and malaise and other symptoms, short duration, low fatality rate. Viral diarrhea in children is a common disease, is an important cause of death in children. Children's diarrhea virus mainly caused by rotavirus (rotavirus), enteric adenovirus (adenovirus), astrovirus (astrovirus, Astv), norovirus (norovirus, NoV)..
Patients with asymptomatic carriers is the main source of infectious viral gastroenteritis. The fecal oral route transmission is viral gastroenteritis is the most important part, can be spread through the air, it can be through contaminated food or water caused by the crowd or sporadic events. An outbreak of gastroenteritis outbreaks were Hendra and Nipah viruses in the world in 90s, also due to foodborne infection, is due to bat virus contaminated water, fruit, then spread to the horse, pig, human, and finally lead to disease outbreaks.
Astrovirus (Astrovirus, Astv) in 1975 for the first time found in the stool samples of children with gastroenteritis, because the virus particles under the electron microscope is the star, it is named astrovirus. Then all over the world have astrovirus infection reported, it can also cause sporadic epidemic of violence, can also cause iatrogenic infection hospitalized children with diarrhea. Astrovirus detection rate was 2.5%-9.0%, the astrovirus is infant viral gastroenteritis second cause, second only to rotavirus.
Astrovirus can infect people, but also can infect cattle, sheep, pigs, dogs, deer, turkeys, ducks, rats and other animal, lead to different degrees of gastroenteritis. Astrovirus infection, traditionally can be divided into 8 serotypes. Recently, researchers have found that two kinds of star shaped virus in diarrhea patients, astrovirus MLB and astrovirus VA., the study also found that Astv and MLB 1 were astrovirus newly found very similar; Astv VA1 in evolutionary tree with infected sheep and mink astrovirus is most close. There is a certain relationship between the stellate virus suggesting human astrovirus and may the newly discovered animal.
In recent years, were detected astrovirus from Hongkong and Chinese, the bat in the University of Hong Kong research team, the detection rate of 46% and 44.8%; gene analysis showed that bat astrovirus has a high degree of polymorphism, relationship between system of astrovirus infection with the virus may some people. However ecological information about bats star the shape of the virus is still scarce, it is necessary to further expand and different kinds of bats samples in different regions.
Norovirus (Norovirus, NoV) is a group of calicivirus is a virus, the prototype strains of Novak virus in the United States in 1972, Novak was found. Because of this group of viruses isolated easily after variation, in other areas have been discovered and named several similar viruses, collectively referred to as norovirus is a highly pathogenic.NoV the highly contagious stomach virus, can cause acute gastroenteritis, once a person is infected, usually for the development of large-scale infectious group. Unfortunately, due to the high variability of the virus and the lack of suitable animal model, now has not yet developed the ideal NoV vaccine, patients prone to recurrent infections.
The nature of NoV carrying many species, including human, pig, cattle, rat, sea lions, they can lead to different degrees of symptoms. According to the characteristics of NoV gene, were divided into five genetic groups (Gene Group, GG): GG 1, GG II, GG III, GG I GG IV and GGV. five genetic groups, GG, 2 and GGIV can infect humans, GG infection in cattle, GGV is detected in rodent animal.
Although studies have shown that NoV is widely present in a variety of animal body, but epidemiological about animal NoV infection is not clear. NoV in different animal rate in infected animal, experimental study found that mouse class NoV North America infection rate as high as 22.1%, the British found cattle in 11% cases of diarrhea caused by NoV infection ratio but, pig NoV detection rate is relatively low, in the detection of Japanese pig GG II NoV rate was only 0.35% in Holland, the detection rate was 2.0%.
Although there is no evidence that the animal NoV can infect humans, but molecular analysis revealed that NoV animal and human NoV strains were closely related to each other, especially the swine NoV strain can be classified into the same group of genes with human NoV strains (GG II). Recent studies have found that the antibody against porcine is common in human NoV this, to further strengthen the animal may play the role of a human NoV storage hypothesis. In addition, the study also found that shellfish animal can simultaneously carry human and animal NoV NoV, suggesting a possible new gene recombinant strains. Recently, people in Canada are very close to the sequence detected in porcine NoV and bovine NoV sequences and NoVGG II.4 strain. Therefore, the future may have to re evaluate the risk possibility of the spread of NoV species and zoonoses.
In 2009, Wuhan virus Institute bats detected another cause diarrhea virus, enteric adenovirus, the detection rate was 8% (28/350), gene analysis showed that bat adenovirus also has high genetic diversity, suggesting that bats may have the ability to carry a series of highly variable virus research has found the star. Rotavirus and adenovirus two diarrhea related virus in bats, a bat is carrying NoV, yet see the reports in the literature, so we can not help but doubt whether bats carrying a outbreak of NoV. in height based NoV due to variability and multiple hosts, as the more than 2 species of mammals (1000 kinds) a bat is likely to carry the virus, or even their host.
Therefore, in order to further understand the different regions of different types of bats astrovirus the situation at the end of its relationship with human astrovirus infection, while understanding in these areas are carrying bats norovirus, this study has not been reported previously in Hunan, parts of Hainan for the scene, the local bats in the investigation of astrovirus and norovirus.
research method
Collection and processing of 1. samples
From July 2007 to July 2008, in Yueyang, Shaoyang, Haikou three areas of long-term monitoring of bats from 4 bats natural habitat in the scene to capture the bat, number, please bat research experts were identified. Samples were collected two hours, including rectal swab samples and cut from rectum tissue samples stored in water. 200 L RNAlater storage solution in freezing tube, placed in the ice box sealing foam when in the day back to the lab, put in the -80 C refrigerator storage, to be inspected.
Detection and identification of 2. bats virus 2.1 stellate viruses
1. Semi nested PCR screening sample
Referring to the literature, for the highly conserved RdRp gene of the stellate virus, we designed and synthesized 5 primers for nested PCR, including 4 primers and 1 common primers. Nested PCR was used to detect the samples.
Sequence identification and analysis
The positive PCR products were excised, purified and recovered, and then sent to the company for sequencing. In case of positive PCR products with very weak, then do cloning, then after inoculation of bacteria feeding to DNA sequencing.
The sequencing results into GenBank BLAST software for analysis of online source, and then download the related nucleic acid sequences in GenBank, use Clastal_X software to analyze the multiple sequence alignment of sequences to be analyzed, and the nucleic acid sequence similarity was calculated by DNAstar software. Using MEGA4.1 software, were analyzed by Neighbor-Joining method to construct phylogenetic trees.
2.2 norovirus
(1) TaqMan real-time PCR detection of geno type Nuo virus (GG II NoV)
The specific primers and probes used to detect GG II NoV were synthesized with reference to the literature, and the TaqMan real-time PCR method was used to detect the primers.
(2) SYBR Green real-time PCR detection of norovirus
Two pairs of universal primers P289/P290 and 12Y/13I were synthesized with reference to the literature, and the specimens were detected by SYBR Green real-time PCR method respectively.
GGV type NoV specific primer detection specimens
GGV type NoV primers were detected by reference to the literature, and the specimens were detected by the common RT-PCR method.
Fourth, the suspicious and positive PCR products obtained from common RT-PCR were cut, purified, recovered, then sent to the company for sequencing. The suspicious positive PCR products detected by TaqMan real-time PCR were sent to the company for cloning and identification.
3. quality control
(1) the experimental materials such as the liquid gun head, the cryopreservation tube and the EP tube are all disposable supplies.
(2) all the containers and reagents used in the RNA extraction and reverse transcriptase experiment were treated with DEPC water, high temperature and high pressure treatment, and dried to remove RNA enzyme pollution.
3. Wear masks, gloves and hats in the whole process, and operate in the asepsis room biological safety cabinet to prevent the reagent and the experimental process from being polluted.
4. Positive control was strictly set up in the experimental process, negative control and blank control.
Result
1 basic situation of bats
In this study, 321 bat species were collected from four monitoring sites in two provinces of Hunan and Hainan, involving 2 bat species, 4 bat families, 9 species of bats. Altogether 321 specimens of bat feces were prepared, including 59 59 anal swabs and 262 262 rectal samples.
Detection of 2 stellate viruses
The nested RT-PCR detected 29 positive samples, the total positive rate was 9.03% (29/321), in 9 apparently healthy bats were found in 3 species of bats carrying astrovirus, positive bat species including m.schreibersii (22/187), yellow bat (6/38) and brown (1/59), with yellow fruit bats the highest detection rate.
The star virus RdRp gene to construct phylogenetic tree of amino acid sequence part analysis shows that bat astrovirus in this study not only to detect astrovirus differences and from other types of large, but also have great differences. However, there are 5 bat astrovirus and sheep astrovirus (ovine Astv) mink astrovirus (mink, Astv) and a novel human astrovirus Astv VA in evolutionary tree is most similar to the sequence similarity of 51.6%-63.1%. and 8 strains from Shaoyang region in the same cave m.schreibersii detected astrovirus gathered on a small branch, their amino acid sequence similarity to 97%-99%.
Detection of 3 norovirus
In this study, TaqMan real-time PCR, SYBR Green real-time PCR and ordinary RT-PCR were used in three experimental methods. Multiple pairs of primers were used to screen the carrying condition of GG type I - IV NoV in the stool samples.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

【参考文献】