基于宏基因组学的动物病毒侦测方法的建立
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摘要
极其多样性的病毒广泛存在于我们的周围环境和动物体内，其中很多病毒具有感染性。发现未知病毒常常受制于病毒常规检测技术的局限性。有很大一部分病毒很难通过细胞培养方法进行分离；与宿主细胞相比，病毒及其核酸的含量极低也是一大问题，而且有些病毒基因组序列信息缺乏或不足，因此很多病毒通过常规病毒分离和PCR方法难以被检测到。
病毒宏基因组学（Viralmetagenomics）是研究特定环境中的病毒群落的一项技术。此方法不依赖已知核酸序列和病毒分离培养，弥补了传统检测方法的不足，在未知病毒的探测方面表现出了极大的优势，已经在诸多领域得到广泛的应用。
本研究旨在应用病毒宏基因组学的理念，结合新型分子诊断技术，构建未知病毒检测技术平台。首先，用除菌过滤及核酸酶消化的方法处理样本，依靠病毒粒子相对较小、其核酸受核衣壳保护的特点，尽可能地去除样本中的宿主内源核酸。将处理后的样本一分为二，分别提取DNA和RNA，均使用5′端带有一段通用序列的随机引物进行双链cDNA和双链DNA合成，随后用这一通用序列作为引物进行随机PCR扩增，PCR产物经限制性内切酶EcoRV除去引物通用序列，回收600~1,500bp的核酸片段插入克隆载体，然后经测序及序列分析，揭示病毒的核酸信息，从而达到对未知病毒检测的目的。我们以猪瘟病毒（CSFV）细胞培养物作为检测对象，建立了RNA病毒检测技术平台，在测序的15条核酸序列中有4条为CSFV序列，序列总长度为1,680bp，占CSFV基因组的13.7%；以猪圆环病毒2型（PCV2）感染猪病料作为检测对象，建立了DNA病毒检测技术平台，在测序的20条核酸序列中有12条为PCV2序列，序列总长度834bp，占PCV2基因组的47.2%；利用此检测技术平台分析一不明病毒细胞培养物，通过测序和序列分析显示，56条序列中有26条为副流感病毒5型（PIV5）同源序列，这些序列位于PIV5的NP、M、F、HN和L基因，覆盖了其基因组全长的16.4%。
本研究建立的基于病毒宏基因组学的未知病毒的检测方法突破了传统检测方法的障碍，既能检测已知病毒又能检测未知病毒，既适用于DNA病毒又适用于RNA病毒，为新发、突发感染性疾病的诊断奠定了基础。
There exist extreme varieties of viruses in the environment and animals, some of which are infectious agents. However, many unknown viruses are barely detected by means of conventional tests, such as virus isolation and PCR assay, due to the following reasons:most of these viruses could hardly be isolated by cell culture; the amounts of viruses and their nucleic acids are relatively low compared to their host cells; the genomic sequence information of some viruses is absent or insufficient in available databases.
Viral metagenomics is a technology used to study a population of viruses in a specific circumstance. This method specially emphasizes on sequence-independent amplification and thus eliminates the process of in vitro viral amplification, which compensates the deficiency of classical methods and shows great advantages in identifying unknown viruses, and it has been extensively utilized in novel virus detection among others. The key to metagenomics-based viral discovery is reducing background nucleic acids.
The aim of this study was to develop an unknown virus detection technology platform based on viral metagenomics in combination with molecular diagnosis. Firstly, filtration and enzymatic removal of cellular nucleic acids were performed to reduce background nucleic acids. Next, DNA and RNA were extracted respectively. RNA reverse transcription and double-stranded cDNA/DNA synthesis were performed with a primer with a random hexanucleotide at its3'-end and a defined sequence at the5'-end. Subsequent random PCR was carried out with the defined5'-end of the initial primer. The amplified products were purified and digested with restriction enzyme EcoRV to remove the amplification primer. Products were then separated on an agarose gel and fragments between600and1,500bp in length were excised and extracted. Purified PCR products were ligated to the pSIMPLE18EcoRV/BAP Vector and introduced into chemically competent E coli TOP10cells. Screened positive clones were sequenced to acquire information of possible viruses. We built analysis technology platform for RNA viruses by testing classical swine fever virus (CSFV)-infected cells, and4out of15sequences were highly homologous to CSFV Analysis of a tissue sample infected with porcine circovirus type2(PCV2) showed that12out of20sequences were homologous to PCV2. We amplified16.4%sequences of the simian parainfluenza virus type5genome from a cell culture of unknown virus using the technology platform developed in this study.
The results indicated that the developed method was suitable for detection of DNA and RNA viruses, and it also has potential for universal detection of known or unknown viruses. It might play a very important role in diagnosis of emerging infectious diseases.
病毒宏基因组学（Viralmetagenomics）是研究特定环境中的病毒群落的一项技术。此方法不依赖已知核酸序列和病毒分离培养，弥补了传统检测方法的不足，在未知病毒的探测方面表现出了极大的优势，已经在诸多领域得到广泛的应用。
本研究旨在应用病毒宏基因组学的理念，结合新型分子诊断技术，构建未知病毒检测技术平台。首先，用除菌过滤及核酸酶消化的方法处理样本，依靠病毒粒子相对较小、其核酸受核衣壳保护的特点，尽可能地去除样本中的宿主内源核酸。将处理后的样本一分为二，分别提取DNA和RNA，均使用5′端带有一段通用序列的随机引物进行双链cDNA和双链DNA合成，随后用这一通用序列作为引物进行随机PCR扩增，PCR产物经限制性内切酶EcoRV除去引物通用序列，回收600~1,500bp的核酸片段插入克隆载体，然后经测序及序列分析，揭示病毒的核酸信息，从而达到对未知病毒检测的目的。我们以猪瘟病毒（CSFV）细胞培养物作为检测对象，建立了RNA病毒检测技术平台，在测序的15条核酸序列中有4条为CSFV序列，序列总长度为1,680bp，占CSFV基因组的13.7%；以猪圆环病毒2型（PCV2）感染猪病料作为检测对象，建立了DNA病毒检测技术平台，在测序的20条核酸序列中有12条为PCV2序列，序列总长度834bp，占PCV2基因组的47.2%；利用此检测技术平台分析一不明病毒细胞培养物，通过测序和序列分析显示，56条序列中有26条为副流感病毒5型（PIV5）同源序列，这些序列位于PIV5的NP、M、F、HN和L基因，覆盖了其基因组全长的16.4%。
本研究建立的基于病毒宏基因组学的未知病毒的检测方法突破了传统检测方法的障碍，既能检测已知病毒又能检测未知病毒，既适用于DNA病毒又适用于RNA病毒，为新发、突发感染性疾病的诊断奠定了基础。
There exist extreme varieties of viruses in the environment and animals, some of which are infectious agents. However, many unknown viruses are barely detected by means of conventional tests, such as virus isolation and PCR assay, due to the following reasons:most of these viruses could hardly be isolated by cell culture; the amounts of viruses and their nucleic acids are relatively low compared to their host cells; the genomic sequence information of some viruses is absent or insufficient in available databases.
Viral metagenomics is a technology used to study a population of viruses in a specific circumstance. This method specially emphasizes on sequence-independent amplification and thus eliminates the process of in vitro viral amplification, which compensates the deficiency of classical methods and shows great advantages in identifying unknown viruses, and it has been extensively utilized in novel virus detection among others. The key to metagenomics-based viral discovery is reducing background nucleic acids.
The aim of this study was to develop an unknown virus detection technology platform based on viral metagenomics in combination with molecular diagnosis. Firstly, filtration and enzymatic removal of cellular nucleic acids were performed to reduce background nucleic acids. Next, DNA and RNA were extracted respectively. RNA reverse transcription and double-stranded cDNA/DNA synthesis were performed with a primer with a random hexanucleotide at its3'-end and a defined sequence at the5'-end. Subsequent random PCR was carried out with the defined5'-end of the initial primer. The amplified products were purified and digested with restriction enzyme EcoRV to remove the amplification primer. Products were then separated on an agarose gel and fragments between600and1,500bp in length were excised and extracted. Purified PCR products were ligated to the pSIMPLE18EcoRV/BAP Vector and introduced into chemically competent E coli TOP10cells. Screened positive clones were sequenced to acquire information of possible viruses. We built analysis technology platform for RNA viruses by testing classical swine fever virus (CSFV)-infected cells, and4out of15sequences were highly homologous to CSFV Analysis of a tissue sample infected with porcine circovirus type2(PCV2) showed that12out of20sequences were homologous to PCV2. We amplified16.4%sequences of the simian parainfluenza virus type5genome from a cell culture of unknown virus using the technology platform developed in this study.
The results indicated that the developed method was suitable for detection of DNA and RNA viruses, and it also has potential for universal detection of known or unknown viruses. It might play a very important role in diagnosis of emerging infectious diseases.
引文
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8.赵建军.鉴别猪瘟病毒野毒株和兔化弱毒疫苗株的复合实时荧光定量RT-PCR方法的建立与评价.[硕士学位论文].北京：中国农业科学院,2007.
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2.楚雍烈,杨娥.宏基因组及其技术的研究进展.西安交通大学学报,2008,29(6)：601-608.
3.黄微,徐顺来.中国养猪业现状与发展方向.畜禽业,2011,269：4-8.
4.钱莉莉,陈少欣,史炳照.宏基因组学在新型生物催化剂开发中的研究进展.微生物学杂志,2006,26(4)：83-87.
5.单同领.病毒宏基因组学分析儿童和猪肠道病毒群落及23株病毒的初步研究.[博士学位论文].上海：上海交通大学,2011.
6.孙玉兰,李德新.新病毒鉴定的分子生物学技术.病毒学报,2011,27(2)：170-175.
7.吴凡,贺争鸣,邢瑞昌.猴副流感病毒(SV5)研究进展.实验动物科学与管理,2006,23(2)：54-57.
8.赵建军.鉴别猪瘟病毒野毒株和兔化弱毒疫苗株的复合实时荧光定量RT-PCR方法的建立与评价.[硕士学位论文].北京：中国农业科学院,2007.
9. Allander T, Tammi MT, Eriksson M, et al. Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci USA,2005,102(36):12891-12896.
10. Attoui H, Biagini P, Stirling J, et al. Sequence characterization of Ndelle virus genome segments1,5,7,8, and10:evidence for reassignment to the genus Orthoreovirus, family Reoviridae. Biochem Biophys Res Commun,2001,287(2):583-588.
11. Blomstrom AL, Widen F, Hammer AS, et al. Detection of a novel astrovirus in brain tissue of mink suffering from Shaking Mink Syndrome by use of viral metagenomics. J Clinical Microbiol,2010,48(12):4392-4396.
12. Bowden TR, Westenberg M, Wang LF, et al. Molecular characterization of Menangle virus, a novel paramyxovirus which infects pigs, fruitbats, and humans. Virology,2001,283:358-373.
13. Bowler LD. Representational difference analysis of cDNA. Methods Mol Med,2004,94:49-66.
14. Breitbart M, Hewson I, Felts B, et al. Metagenomic analyses of an uncultured viral community from human feces. J Bacteriol,2003,85(20):6220-6223.
15. Breitbart M, Salamon P, Andresen B, et al. Genomic analysis of uncultured marine viral communities. Proc Natl Acad Sci USA,2002,99(22):14250-14255.
16. Challoner PB, Smith KT, Parker JD, et al. Plaque-associated expression of human herpesvirus6in multiple selerosis. Proc Natl Acad Sci USA,1995,92:7440-7444.
17. Chang Y, Cesarman E, Pessin MS, et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science,1994,266(5192):1865-1869.
18. Chatziandreou N, Stock N, Young D, et al. Relationships and host range of human, canine, simian and porcine isolates of simian virus5(parainfluenza virus5). J Gen Virol,2004,85:3007-3016.
19. Chiu CY, Greninger AL, Kanada K, et al. Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections. Proc Natl Acad Sci USA,2008,105(37):14124-14129.
20. Choo QL, Kuo G, Weiner AJ, et al. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science,1989,244(4902):359-362.
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