本文選題：獼猴 + 高通量測序��； 參考：《中國疾病預(yù)防控制中心》2017年碩士論文

【摘要】：背景:近年來,新發(fā)突發(fā)傳染病屢次發(fā)生,對人類健康造成了嚴重的威脅,已成為目前全球最重要的公共衛(wèi)生問題之一。2003年的SARS、2005年的禽流感、2009年的甲型H1N1大流感、2012年中東的MERS、2013年我國H7N9禽流感,以及2014年西非埃博拉等新發(fā)突發(fā)傳染病都呈現(xiàn)出一個共同特點,即病毒都是動物源性。隨著人類活動范圍的擴大,人類與野生動物的活動關(guān)系越來越親近,動物病毒感染人類的機會隨之增加。據(jù)報道70%以上的新發(fā)突發(fā)傳染病都是經(jīng)過動物傳播給人類的,因此動物源性病毒的研究是疾病預(yù)防控制的重要內(nèi)容。近年來,許多具有高致病性的病毒在非人靈長類動物中相繼被發(fā)現(xiàn)。非人靈長類動物可以作為這些病毒的重要儲存宿主,并將其傳播給人類,如人類免疫缺陷病毒、埃博拉病毒和馬爾堡病毒等。病毒宏基因組學是一種可基于隨機PCR和高通量測序技術(shù),并對臨床或者環(huán)境標本中病毒進行系統(tǒng)的分析和鑒定的方法。獼猴是自然界中最常見的一種猴,分布廣泛,且生理上與人類較接近。因此,研究獼猴所攜帶的病毒譜,對非人靈長類動物的保護以及人類健康的防護具有極其重要的意義。方法:收集我國廣西自然保護區(qū)龍虎山野生獼猴糞便標本,利用建立好的高通量測序前樣品處理方法進行樣品處理;構(gòu)建病毒DNA測序文庫、進行Hiseq高通量測序;利用生物信息分析平臺對測序后海量數(shù)據(jù)進行病毒宏基因組學分析,獲得獼猴糞便的病毒譜;對一些潛在意義的病毒,進行PCR驗證、分子流行病學調(diào)查、全基因組的擴增和細胞分離等深入研究,以獲得該病毒的流行動態(tài)和遺傳進化關(guān)系等病原學特征。結(jié)果:本研究經(jīng)高通量測序后一共獲得340,899,838條序列,其中有1,126,694條序列被注釋到病毒。序列分析發(fā)現(xiàn)這些病毒序列可以劃分為53個病毒科和104個病毒屬,包括12個脊椎動物病毒科(包含31個病毒屬)、14個無脊椎動物病毒科和18個植物病毒科以及8個噬菌體科。本研究發(fā)現(xiàn)的脊椎動物病毒主要集中在小RNA病毒(53%)、小雙節(jié)病毒(28%)、環(huán)狀病毒(11%)和細小病毒(6%)等。對這些脊椎動物病毒序列進行深入分析,結(jié)果發(fā)現(xiàn)獼猴中存在多個新型病毒,主要包括與人星狀病毒MLB和VA相近的星狀病毒,氨基酸同源性為75-90%;與J組猴腸道病毒具有59%的氨基酸同源性的新型腸道病毒;與B組猴薩佩羅病毒較為接近的新型薩佩羅病毒;與小RNA病毒Falcovirus/Hepotovirus/Tremovirus 具有 24-27%氨基酸同源性的新型小 RNA 病毒;與人博卡病毒具有最高同源性的細小病毒,氨基酸相似度為51-100%;人諾如病毒GII.17和GI.3,核酸同源性高達98%。本研究挑選了腸道病毒(Enterovirus,EV)(命名SEV-gx)和同源性極低的小RNA病毒(命名MobovirusA)進行了深入研究。SEV-gx全基因組長度為7,367個核苷酸。高達10%的陽性檢測率提示該病毒能夠廣泛感染當?shù)孬J猴。27個SEV-gx VP1序列所呈現(xiàn)出的100%核酸同源性,提示該病毒在當?shù)孬J猴中能夠穩(wěn)定地傳播。SEV-gx具有典型的EV基因組結(jié)構(gòu)和特征性結(jié)構(gòu)域。序列比對顯示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蛋白進化樹分析顯示SEV-gx在腸道病毒屬中形成一個獨立進化枝。因此,本研究發(fā)現(xiàn)的新病毒應(yīng)被劃為腸道病毒屬中的一個新的病毒種(建議命名EV-K)。Mobovirus A基因組全長為8,325個核苷酸。序列比對顯示Mobovirus A和小RNA病毒Falcovirus/Tremovirus/Hepatovirus具有較低的同源性,氨基酸相似度分別為P122%、P224和P324%。同時,P1、2C和3D區(qū)氨基酸進化樹分析顯示Mobovirus A在Picornaviriade中形成一個獨立進化枝,與Falcovirus/Tremovirus/Hepatovirus在進化關(guān)系中最接近。基因組結(jié)構(gòu)分析發(fā)現(xiàn)Mobovirus A具有典型的小 RNA病毒科的基因組成特征,即5,UTR-VP2-VP0-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3'UTR,類似于 Falcovirus/Tremovirus/Hepatovirus。蛋白分析顯示Mobovirus A具有小RNA病毒典型的結(jié)構(gòu)域。根據(jù)國際病毒學分類委員會建議的小RNA病毒屬分類的原則,本研究發(fā)現(xiàn)的新病毒應(yīng)該被歸納為Picornaviriade中新的病毒屬(建議命名為Monkey-borne-virus,Mobovirus)。結(jié)論:本研究通過病毒宏基因組學方法深入了解到了我國廣西龍虎山野生獼猴糞便所攜帶的病毒譜情況,其中發(fā)現(xiàn)了許多新病毒的存在,如小RNA病毒(新型腸道病毒)、類似人星狀病毒、人博卡樣病毒和人諾如病毒等,極大地豐富了我國及全球野生動物攜帶病毒數(shù)據(jù)庫。本研究在我國野生獼猴糞便中發(fā)現(xiàn)了新的小RNA病毒屬和腸道病毒種,這將有利于人們對小RNA病毒的遺傳多樣性和進化關(guān)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.

【學位授予單位】：中國疾病預(yù)防控制中心
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R373

【參考文獻】

相關(guān)期刊論文 前1條

1 Marcello Guido;Maria Rosaria Tumolo;Tiziano Verri;Alessandro Romano;Francesca Serio;Mattia De Giorgi;Antonella De Donno;Francesco Bagordo;Antonella Zizza;;Human bocavirus: Current knowledge and future challenges[J];World Journal of Gastroenterology;2016年39期

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本文編號：1797343