Influenza A virus molecular virology techniques.
Influenza A virus molecular virology techniques.
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DOI：
10.1007/978-1-61779-621-0_11
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发表时间：
2012-01-01
期刊：
影响因子：
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通讯作者：
Wentworth, David E
中科院分区：
文献类型：
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作者：
Zhou, Bin;Wentworth, David E
用于基因组分析和创造重组病毒的分子生物学技术是我们努力了解和抗击甲型流感病毒的关键工具。这些分子病毒学方法被用于诊断学、基础研究、分子流行病学、生物信息学和疫苗开发。大多数用于研究这种节段式负义RNA病毒的技术都是从病毒或受感染的细胞中提纯RNA，并将其转化为cDNA，然后转化为dsDNA，并使用逆转录和聚合酶链式反应(RTPCR)相结合的方法来扩增dsDNA。RT-PCR扩增片段可以被探测、测序或克隆到各种载体中，用于进一步分析，并通过基于质粒的反向遗传学创造重组甲型流感病毒。为了加快扩增和克隆过程，我们开发了多片段RT-PCR(M-RTPCR)技术，该技术可以在一次反应中有效地扩增甲型流感病毒的8个基因组RNA片段(VRNAs)，与病毒株无关。M-RTPCR扩增片段是核苷酸序列分析和将全长vRNA克隆到为蛋白质表达或反向遗传学而设计的载体中的理想选择。因此，我们还开发了改进的反向遗传学质粒，旨在使用基于重组的技术快速克隆M-RTPCR产物或其他全长vRNA扩增片段。M-RTPCR和基于重组的克隆的结合赋予了分析和拯救任何甲型流感病毒株/亚型的敏感性、速度、保真度和灵活性，而不需要体外繁殖。本章中描述的具体主题包括高质量病毒RNA的纯化、使用两种不同的M-RTPCR方案进行基因组扩增、对vRNA扩增片段进行测序，以及将vRNA扩增片段克隆到我们改良的反向遗传学质粒中，或商业上可获得的质粒中。
Molecular biological techniques for genomic analysis and for creation of recombinant viruses are critical tools in our efforts to understand and combat influenza A viruses. These molecular virology approaches are used in diagnostics, basic research, molecular epidemiology, bioinformatics, and vaccine development. The majority of the techniques used to study this segmented negative-sense RNA virus begin by purifying RNA from the virus, or infected cells, and converting it to cDNA, then to dsDNA, and amplifying that dsDNA using reverse transcription in combination with the polymerase chain reaction (RTPCR). The RTPCR amplicons can be probed, sequenced, or cloned into a variety of vectors for further analysis and to create recombinant influenza A viruses by plasmid-based reverse genetics. To accelerate the amplification and cloning process, we developed multi-segment-RTPCR (M-RTPCR) techniques that efficiently amplify the eight genomic viral RNA segments (vRNAs) of influenza A virus in a single reaction, irrespective of the virus strain. The M-RTPCR amplicons are ideal for nucleotide sequence analysis and cloning full-length vRNAs into plasmids or other vectors designed for protein expression or reverse genetics. Therefore, we also developed modified reverse-genetics plasmids that are designed to rapidly clone M-RTPCR products, or other full-length vRNA amplicons, using recombination-based techniques. The combination of M-RTPCR and recombination-based cloning confers sensitivity, speed, fidelity, and flexibility to the analysis and rescue of any strain/subtype of influenza A virus, without the need for in vitro propagation. The specific topics described in this chapter include purification of high-quality viral RNA, genomic amplification using two different M-RTPCR schemes, sequencing vRNA amplicons, and cloning vRNA amplicons into our modified reverse-genetics plasmids, or commercially available plasmids.