本文選題：東北虎　切入點(diǎn)：病毒宏基因組學(xué)　出處：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：病毒是地球上最豐富的物種之一,很多病毒嚴(yán)重威脅著人類和動物的安全,近年來,新發(fā)、突發(fā)病毒性傳染病陸續(xù)出現(xiàn),鑒于其高度的不可知性,突發(fā)性,傳播迅速,病死率高,危害大的特點(diǎn),極易引起群體性恐慌和影響社會穩(wěn)定。近年來興起的病毒宏基因組學(xué)(viral metagenomics)不依賴傳統(tǒng)的病毒分離,它突破了傳統(tǒng)病毒學(xué)研究方法的局限,直接針對環(huán)境中所有的病毒核酸進(jìn)行研究,能夠快速的鑒定出環(huán)境中病毒群落組成,成為一種發(fā)現(xiàn)新病毒,監(jiān)控病毒變異的有力手段。虎作為大型肉食動物,位于食物鏈的頂端,各種病毒包括犬瘟熱病毒,貓細(xì)小病毒,犬副流感病毒,貓杯狀病毒,犬腺病毒,甚至禽流感病毒都已經(jīng)在虎體內(nèi)發(fā)現(xiàn),這些病毒不僅威脅著虎的生存,而且有些還威脅著人類的健康,尤其現(xiàn)代社會,人類與虎的接觸機(jī)會越來越多,對虎病毒性疾病的研究變得很有必要,而目前還未有虎源消化道病毒群落系統(tǒng)研究的報(bào)道。本研究應(yīng)用病毒宏基因組學(xué)技術(shù),對東北虎源消化道病毒進(jìn)行探究。首先,隨機(jī)收集東北虎糞便樣品,對糞便進(jìn)行PBS稀釋,通過高速離心的方法去除無關(guān)雜質(zhì)成分,通過超速離心方法對病毒粒子濃縮,然后用核酸酶消化方法對病毒粒子外核酸進(jìn)行分解。抽提核酸后應(yīng)用不依賴序列的單引物擴(kuò)增方法(SISPA)對病毒核酸進(jìn)行擴(kuò)增,對擴(kuò)增后濃縮純化產(chǎn)物進(jìn)行Illumina Hi Seq2000高通量測序,共得到3200,000,000條讀長(Reads),并拼接出6766個序列重疊群(Contigs),序列重疊群全長為8,451,561 bp。經(jīng)過生物信息學(xué)分析后,其中涵蓋病毒序列主要對應(yīng)到:貓細(xì)小病毒、貓杯狀病毒、犬細(xì)小病毒、豬博卡病毒、哺乳動物呼腸孤病毒、藍(lán)舌病病毒、流感病毒、貂腸炎病毒、雞傳染性貧血病毒等。本研究分離得到一株貓細(xì)小病毒HRB T2014和一株貓杯狀病毒TFHLJ-8,對宏基因組測序結(jié)果進(jìn)行了驗(yàn)證。對HRB T2014的VP2基因序列同源性分析表明,其與下載的參考毒株的相似性為98.1%~99.9%之間,相似性最高的是國內(nèi)虎源貓細(xì)小病毒FJ405225株和HT-374株,相似性最低的是犬細(xì)小病毒cpv-nj01-06株,系統(tǒng)進(jìn)化樹分析中,HRB T2014處于虎源貓細(xì)小病毒分支,HRB-T2014株全基因序列分析表明,其與參考毒株的相似性在98.6%-99.3%之間。對TFHLJ-8全基因序列分析表明,其與其他參考毒株的相似性在76.3%-92%之間,相似性最高的為URB株,相似性最低的為GD株。本研究對東北虎消化道源病毒進(jìn)行了病毒宏基因組學(xué)分析,測序結(jié)果得到了貓細(xì)小病毒,貓杯狀病毒、犬細(xì)小病毒、藍(lán)舌病病毒,哺乳動物呼腸孤病毒,流感病毒等病毒序列;并對貓細(xì)小病毒和貓杯狀病毒成功進(jìn)行了驗(yàn)證;分離得到了一株貓細(xì)小病毒HRB T2014,分析表明此細(xì)小病毒有可能是犬細(xì)小病毒和貓細(xì)小病毒的重組株,同時分離到一株貓杯狀病毒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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.93

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本文編號：1679328