东北虎源消化道病毒宏基因组学分析

发布时间：2018-03-29 03:21

本文选题：东北虎　切入点：病毒宏基因组学　出处：《中国农业科学院》2015年硕士论文

【摘要】：病毒是地球上最丰富的物种之一,很多病毒严重威胁着人类和动物的安全,近年来,新发、突发病毒性传染病陆续出现,鉴于其高度的不可知性,突发性,传播迅速,病死率高,危害大的特点,极易引起群体性恐慌和影响社会稳定。近年来兴起的病毒宏基因组学(viral metagenomics)不依赖传统的病毒分离,它突破了传统病毒学研究方法的局限,直接针对环境中所有的病毒核酸进行研究,能够快速的鉴定出环境中病毒群落组成,成为一种发现新病毒,监控病毒变异的有力手段。虎作为大型肉食动物,位于食物链的顶端,各种病毒包括犬瘟热病毒,猫细小病毒,犬副流感病毒,猫杯状病毒,犬腺病毒,甚至禽流感病毒都已经在虎体内发现,这些病毒不仅威胁着虎的生存,而且有些还威胁着人类的健康,尤其现代社会,人类与虎的接触机会越来越多,对虎病毒性疾病的研究变得很有必要,而目前还未有虎源消化道病毒群落系统研究的报道。本研究应用病毒宏基因组学技术,对东北虎源消化道病毒进行探究。首先,随机收集东北虎粪便样品,对粪便进行PBS稀释,通过高速离心的方法去除无关杂质成分,通过超速离心方法对病毒粒子浓缩,然后用核酸酶消化方法对病毒粒子外核酸进行分解。抽提核酸后应用不依赖序列的单引物扩增方法(SISPA)对病毒核酸进行扩增,对扩增后浓缩纯化产物进行Illumina Hi Seq2000高通量测序,共得到3200,000,000条读长(Reads),并拼接出6766个序列重叠群(Contigs),序列重叠群全长为8,451,561 bp。经过生物信息学分析后,其中涵盖病毒序列主要对应到:猫细小病毒、猫杯状病毒、犬细小病毒、猪博卡病毒、哺乳动物呼肠孤病毒、蓝舌病病毒、流感病毒、貂肠炎病毒、鸡传染性贫血病毒等。本研究分离得到一株猫细小病毒HRB T2014和一株猫杯状病毒TFHLJ-8,对宏基因组测序结果进行了验证。对HRB T2014的VP2基因序列同源性分析表明,其与下载的参考毒株的相似性为98.1%~99.9%之间,相似性最高的是国内虎源猫细小病毒FJ405225株和HT-374株,相似性最低的是犬细小病毒cpv-nj01-06株,系统进化树分析中,HRB T2014处于虎源猫细小病毒分支,HRB-T2014株全基因序列分析表明,其与参考毒株的相似性在98.6%-99.3%之间。对TFHLJ-8全基因序列分析表明,其与其他参考毒株的相似性在76.3%-92%之间,相似性最高的为URB株,相似性最低的为GD株。本研究对东北虎消化道源病毒进行了病毒宏基因组学分析,测序结果得到了猫细小病毒,猫杯状病毒、犬细小病毒、蓝舌病病毒,哺乳动物呼肠孤病毒,流感病毒等病毒序列;并对猫细小病毒和猫杯状病毒成功进行了验证;分离得到了一株猫细小病毒HRB T2014,分析表明此细小病毒有可能是犬细小病毒和猫细小病毒的重组株,同时分离到一株猫杯状病毒TFHLJ-8。
[Abstract]:Virus is one of the most abundant species on the earth. Many viruses seriously threaten the safety of human beings and animals. In recent years, new and sudden viral infectious diseases have emerged one after another. Because of its high mortality and great harm, it can easily cause mass panic and affect social stability. In recent years, viral macrogenomics (virogenomics), which does not rely on traditional virus isolation, has broken through the limitations of traditional virology research methods. Direct research on all viral nucleic acids in the environment can quickly identify the composition of the virus community in the environment and become a powerful means of detecting new viruses and monitoring the variation of the virus. The tiger is a large carnivorous animal. At the top of the food chain, viruses including canine distemper virus, cat parvovirus, canine parainfluenza virus, cat calix virus, canine adenovirus, and even avian influenza virus have been found in tigers, which not only threaten the survival of tigers, And some are threatening human health, especially in modern society, where human contact with tigers is increasing, so it is necessary to study tiger viral diseases. However, there is no systematic study on the alimentary tract virus community of Amur tiger. In this study, the virus macrogenomics technique was used to investigate the alimentary tract virus from Amur tiger. Firstly, the fecal samples of Amur tiger were collected randomly and diluted with PBS. The independent impurity is removed by high-speed centrifugation, and the virus particles are concentrated by ultracentrifugation. After the nucleic acid was extracted, the nucleic acid was amplified by single sequence-independent primer amplification method (sispa), and the purified product was amplified by Illumina Hi Seq2000 high-throughput sequencing. A total of 3200000000 Readsmongs were obtained and 6766 overlapped groups were constructed, the total length of which was 8451561 BP. After bioinformatics analysis, the sequences of them were mainly corresponding to: cat parvovirus, cat calix virus, canine parvovirus, and canine parvovirus. Porcine Boca virus, mammalian reovirus, blue-tongue virus, influenza virus, mink enteritis virus, In this study, a cat parvovirus HRB T2014 and a cat calix virus TFHLJ-8 were isolated, and the results of macro genome sequencing were verified. The homology of VP2 gene sequence of HRB T2014 was analyzed. The similarity between it and the downloaded reference strain was 98.1% and 99.9% respectively. The highest similarity was found in domestic tiger source cat parvovirus FJ405225 and HT-374 strains, and the lowest similarity was found in canine parvovirus cpv-nj01-06 strain. In phylogenetic tree analysis, the similarity between HRB-T2014 and the reference strain was 98.6- 99.3%, and the similarity between HRB-T2014 and other reference strains was 76.3- 92%. The highest similarity was found in URB strain, and the lowest in GD strain. In this study, virus macrogenomics analysis of Amur tiger digestive tract virus was carried out. The results of sequencing showed that cat parvovirus, cat calix virus, canine parvovirus, bluetongue virus were obtained. Mammalian reovirus and influenza virus were sequenced, and the cat parvovirus and cat calix virus were successfully verified. A cat parvovirus HRB T2014 was isolated. The analysis indicated that the parvovirus might be a recombinant strain of canine parvovirus and cat parvovirus, and a cat calix virus TFHLJ-8 was isolated at the same time.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.93

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