基于宏基因组学的猪群样本病毒探测方法的建立

Metagenomics-based detection of swine viruses

极其多样的病毒广泛存在于我们周围的环境和动物体内,其中很多病毒是人类所未知的,而发现未知病毒常常受制于病毒常规检测技术的局限性。【目的】构建未知病毒检测技术平台。【方法】应用病毒宏基因组学的理念,结合新型分子诊断技术,首先利用过滤和核酸酶处理去除样品宿主核酸干扰,然后随机PCR扩增潜在的病毒宏基因组,最后通过大规模测序及序列分析获取病毒核酸信息。【结果】利用此技术我们对猪瘟病毒(CSFV)细胞培养物和猪圆环病毒2型(PCV2)感染猪病料进行了分析,分别检测到序列总长度1680 bp,占基因组13.7%的CSFV序列和序列总长度834 bp,占基因组47.2%的PCV2序列;利用此检测技术平台研究一未知病原细胞培养物,通过测序和序列分析,结果显示56条序列中有26条为副流感病毒5型(PIV5)同源序列,覆盖了其基因组全长的16.4%;此外,应用本研究建立的方法结合新一代高通量测序,我们在混合的7份病原未知的病猪组织样品中检测到了1.1%的病毒序列,包括CSFV、PCV2、猪细环病毒(TTSuV)、猪bocavirus(PBoV)和人腺病毒6型(Ad6)等的部分基因序列。【结论】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病毒研究方法的缺陷,对于猪群样本中病毒的检测具有较高的敏感性,有望为新发、突发感染性疾病的诊断和监测提供技术支持。

Extreme varieties of viruses exist in the environment and animals,some of which are unknown.However,many unknown viruses are barely detected by means of conventional virus isolation and PCR assay.[Objective] To develop a technology platform for detecting unknown viruses.[Methods] We established the technology based on viral metagenomics in combination with novel molecular diagnostics.The technology is consisted of removal of host nucleic acid,random PCR amplification,large-scale sequencing,and bioinformatics.[Results] The technology was applied to detect classical swine fever virus（CSFV）-infected cells and a tissue sample of a pig infected with porcine circovirus type 2（PCV2）.We amplified 13.7% sequences of CSFV genome and 47.2% those of PCV2 genome,respectively.Moreover,we amplified 16.4% sequences of the simian parainfluenza virus type 5 genome from an unknown virus cell culture using the developed method.In addition,using the developed method combined with the high-throughput sequencing,we detected 1.1% virus sequences,including CSFV,PCV2,torque teno sus virus（TTSuV）,porcine bocavirus（PBoV）and human adenovirus type 6（Ad6） from 7 clinical swine samples of unknown causative agents.[Conclusion] The developed metagenomics-based method showed good sensitivity for detection of both DNA and RNA viruses from diverse swine samples,and has potential for universal detection of known and unknown viruses.It might facilitate the diagnosis of emerging viral diseases.