广西猕猴粪便病毒宏基因组研究

发布时间：2018-04-24 16:15

本文选题：猕猴 + 高通量测序　；参考：《中国疾病预防控制中心》2017年硕士论文

【摘要】：背景:近年来,新发突发传染病屡次发生,对人类健康造成了严重的威胁,已成为目前全球最重要的公共卫生问题之一。2003年的SARS、2005年的禽流感、2009年的甲型H1N1大流感、2012年中东的MERS、2013年我国H7N9禽流感,以及2014年西非埃博拉等新发突发传染病都呈现出一个共同特点,即病毒都是动物源性。随着人类活动范围的扩大,人类与野生动物的活动关系越来越亲近,动物病毒感染人类的机会随之增加。据报道70%以上的新发突发传染病都是经过动物传播给人类的,因此动物源性病毒的研究是疾病预防控制的重要内容。近年来,许多具有高致病性的病毒在非人灵长类动物中相继被发现。非人灵长类动物可以作为这些病毒的重要储存宿主,并将其传播给人类,如人类免疫缺陷病毒、埃博拉病毒和马尔堡病毒等。病毒宏基因组学是一种可基于随机PCR和高通量测序技术,并对临床或者环境标本中病毒进行系统的分析和鉴定的方法。猕猴是自然界中最常见的一种猴,分布广泛,且生理上与人类较接近。因此,研究猕猴所携带的病毒谱,对非人灵长类动物的保护以及人类健康的防护具有极其重要的意义。方法:收集我国广西自然保护区龙虎山野生猕猴粪便标本,利用建立好的高通量测序前样品处理方法进行样品处理;构建病毒DNA测序文库、进行Hiseq高通量测序;利用生物信息分析平台对测序后海量数据进行病毒宏基因组学分析,获得猕猴粪便的病毒谱;对一些潜在意义的病毒,进行PCR验证、分子流行病学调查、全基因组的扩增和细胞分离等深入研究,以获得该病毒的流行动态和遗传进化关系等病原学特征。结果:本研究经高通量测序后一共获得340,899,838条序列,其中有1,126,694条序列被注释到病毒。序列分析发现这些病毒序列可以划分为53个病毒科和104个病毒属,包括12个脊椎动物病毒科(包含31个病毒属)、14个无脊椎动物病毒科和18个植物病毒科以及8个噬菌体科。本研究发现的脊椎动物病毒主要集中在小RNA病毒(53%)、小双节病毒(28%)、环状病毒(11%)和细小病毒(6%)等。对这些脊椎动物病毒序列进行深入分析,结果发现猕猴中存在多个新型病毒,主要包括与人星状病毒MLB和VA相近的星状病毒,氨基酸同源性为75-90%;与J组猴肠道病毒具有59%的氨基酸同源性的新型肠道病毒;与B组猴萨佩罗病毒较为接近的新型萨佩罗病毒;与小RNA病毒Falcovirus/Hepotovirus/Tremovirus 具有 24-27%氨基酸同源性的新型小 RNA 病毒;与人博卡病毒具有最高同源性的细小病毒,氨基酸相似度为51-100%;人诺如病毒GII.17和GI.3,核酸同源性高达98%。本研究挑选了肠道病毒(Enterovirus,EV)(命名SEV-gx)和同源性极低的小RNA病毒(命名MobovirusA)进行了深入研究。SEV-gx全基因组长度为7,367个核苷酸。高达10%的阳性检测率提示该病毒能够广泛感染当地猕猴。27个SEV-gx VP1序列所呈现出的100%核酸同源性,提示该病毒在当地猕猴中能够稳定地传播。SEV-gx具有典型的EV基因组结构和特征性结构域。序列比对显示SEV-gx和EV-J具有最高同源性(P1蛋白:43.0-44.1%相似度;P2蛋白:52.3-55.2%相似度;P3蛋白:61.1-62.7%相似度;2C+3C蛋白:64.0%相似度)。另外,P1和2C以及3D蛋白进化树分析显示SEV-gx在肠道病毒属中形成一个独立进化枝。因此,本研究发现的新病毒应被划为肠道病毒属中的一个新的病毒种(建议命名EV-K)。Mobovirus A基因组全长为8,325个核苷酸。序列比对显示Mobovirus A和小RNA病毒Falcovirus/Tremovirus/Hepatovirus具有较低的同源性,氨基酸相似度分别为P122%、P224和P324%。同时,P1、2C和3D区氨基酸进化树分析显示Mobovirus A在Picornaviriade中形成一个独立进化枝,与Falcovirus/Tremovirus/Hepatovirus在进化关系中最接近。基因组结构分析发现Mobovirus A具有典型的小 RNA病毒科的基因组成特征,即5,UTR-VP2-VP0-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3'UTR,类似于 Falcovirus/Tremovirus/Hepatovirus。蛋白分析显示Mobovirus A具有小RNA病毒典型的结构域。根据国际病毒学分类委员会建议的小RNA病毒属分类的原则,本研究发现的新病毒应该被归纳为Picornaviriade中新的病毒属(建议命名为Monkey-borne-virus,Mobovirus)。结论:本研究通过病毒宏基因组学方法深入了解到了我国广西龙虎山野生猕猴粪便所携带的病毒谱情况,其中发现了许多新病毒的存在,如小RNA病毒(新型肠道病毒)、类似人星状病毒、人博卡样病毒和人诺如病毒等,极大地丰富了我国及全球野生动物携带病毒数据库。本研究在我国野生猕猴粪便中发现了新的小RNA病毒属和肠道病毒种,这将有利于人们对小RNA病毒的遗传多样性和进化关系的理解。
[Abstract]:Background: in recent years, new outbreak of infectious diseases has occurred repeatedly and poses a serious threat to human health. It has become one of the most important public health problems in the world at present.2003 year SARS, avian influenza in 2005, the Great Influenza A (H1N1) influenza in 2009, MERS in 2012 in the Middle East, H7N9 avian influenza in 2013, and Ebola in West Africa in 2014. All the infectious diseases show a common characteristic, that is, the virus is all animal origin. With the expansion of human activities, the relationship between human and wildlife is becoming more and more close, and the opportunity of animal virus infection is increasing. It is reported that more than 70% of the new infectious diseases are transmitted to humans by animals, so the animals are transmitted to humans. Research on the source of the virus is an important part of disease prevention and control. In recent years, many highly pathogenic viruses have been found in nonhuman primates. Nonhuman primates can be used as an important storage host for these viruses and transmit them to humans, such as the human immunodeficiency virus, Ebola virus and Marburg virus. Virus macrogenomics is a method based on random PCR and high throughput sequencing technology to analyze and identify viruses in clinical or environmental specimens. Macaques are the most common species in nature and are widely distributed and closely related to human beings. Therefore, the virus spectrum carried by rhesus monkeys is studied for nonhuman primates. The protection of animals and the protection of human health are of great significance. Methods: collecting the faeces of wild macaque in Mount Longhu of Guangxi natural reserve of China, using a good high throughput sequencing sample processing method to carry out sample processing, construction of virus DNA sequencing library, Hiseq high throughput sequencing, and bioinformatics analysis. The virus genome was analyzed by the platform on the massive data after sequencing, and the virus spectrum of macaque excrement was obtained. Some potential viruses were examined by PCR, molecular epidemiology, whole genome amplification and cell separation, in order to obtain the pathogenic characteristics of the virus's flow and genetic evolution. A total of 340899838 sequences were sequenced by high flux sequencing, of which 1126694 sequences were annotated to the virus. Sequence analysis found that these sequences could be divided into 53 virulates and 104 viruses, including 12 vertebrate viruses (including 31 genera), 14 invertebrate and 18 plant viruses. The vertebrate viruses found in this study are mainly concentrated in small RNA virus (53%), small double segment virus (28%), cyclic virus (11%) and parvovirus (6%). The sequence of these vertebrate viruses is analyzed in depth. The results show that there are several new viruses in rhesus monkeys, which are mainly similar to the human stellate virus MLB and VA. The stellate virus, amino acid homology 75-90%; a new enterovirus with 59% amino acid homology with the J group of the monkey enterovirus; a new Shapero virus that is closer to the B group of monkey Shapero virus; a new small RNA virus with the homology of 24-27% amino acids with the small RNA virus Falcovirus/Hepotovirus/Tremovirus; and the human Boka's disease. The virus has the highest homologous parvovirus, the amino acid similarity is 51-100%, the human norovirus GII.17 and GI.3, the nucleic acid homology is up to 98%.. The study selected the enterovirus (Enterovirus, EV) (named SEV-gx) and the small RNA virus (named MobovirusA) with very low homology (named MobovirusA). The whole genome length of.SEV-gx was 7367 nucleosides. Acid. The positive detection rate of up to 10% suggests that the virus can be widely infected with the 100% nucleic acid homology of the local rhesus monkey.27 SEV-gx VP1 sequence, suggesting that the virus can steadily spread.SEV-gx in the local macaques with a typical EV genome structure and characteristic domain. Sex (P1: 43.0-44.1% similarity; P2 protein: 52.3-55.2% similarity; P3 protein: 61.1-62.7% similarity; 2C+3C protein: 64% similarity). In addition, P1 and 2C and 3D protein evolution tree analysis showed that SEV-gx in the genus enterovirus was formed an independent branch. Therefore, the new virus found in this study should be classified as a new enterovirus. The total length of.Mobovirus A genome was 8325 nucleotides. Sequence alignment showed low homology between Mobovirus A and small RNA virus Falcovirus/Tremovirus/Hepatovirus. The amino acid similarity was P122%, P224 and P324%. respectively. Riade formed an independent branch and was the closest in the evolutionary relationship with Falcovirus/Tremovirus/Hepatovirus. Genomic structure analysis found that Mobovirus A had a typical gene composition of the small RNA family, that is, 5, UTR-VP2-VP0-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3'UTR, similar to the analysis of Falcovirus/Tremovirus/Hepatovirus. protein. It shows that Mobovirus A has a typical domain of small RNA virus. According to the principle of the classification of small RNA viruses proposed by the international virology Classification Committee, the new virus found in this study should be classified as a new virus in Picornaviriade (named Monkey-borne-virus, Mobovirus). Conclusion: This study was conducted through the viral metagenomics side. The method is to understand the virus spectrum of the wild macaque manure in Mount Longhu, Guangxi, China. Among them, many new viruses are found, such as small RNA virus (New enterovirus), human stellate virus, human Boka like virus and human norovirus, which greatly enriched the database of virus carrying virus in our country and the world. A new species of small RNA virus and enterovirus has been found in the feces of wild macaque in China, which will help people understand the genetic diversity and evolutionary relationship of small RNA viruses.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373

【参考文献】