Detection of Molecular Markers of Antiviral Resistance in Influenza A (H5N1) Viruses Using a Pyrosequencing Method
Detection of Molecular Markers of Antiviral Resistance in Influenza A (H5N1) Viruses Using a Pyrosequencing Method
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DOI：
10.1128/aac.01446-08
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发表时间：
2009-03-01
影响因子：
4.9
通讯作者：
Gubareva, Larisa V.
中科院分区：
文献类型：
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作者：
Deyde, Varough M.;Tung Nguyen;Gubareva, Larisa V.
流感病毒对抗病毒药物的耐药性可能在药物治疗后出现，也可能是自然变异造成的。目前有两类靶向M2蛋白（金刚烷胺和金刚乙胺）或神经氨酸酶（NA;奥司他韦和扎那米韦）的抗流感病毒药物获得许可。预计这些药物在控制潜在大流行的早期阶段很重要。在本研究中，我们描述了如何焦磷酸测序方法可用于快速检测建立的分子标记物的M2阻断剂和NA抑制剂的耐药性甲型（H5 N1）流感病毒。靶向M2蛋白中的残基L26、V27、A30、S31和G34进行焦磷酸测序。用于焦磷酸测序分析的NA残基包括已确立的耐药性标志物（H274和N294）以及不太确定的相关性残基（V116、I117、Q136、K150和I222）。设计了一对焦-逆转录（RT）-PCR引物，以允许扩增来自与人类感染相关的各种进化枝/亚进化枝的H5 N1病毒NA基因的约600个核苷酸长的扩增子。通过对从系列稀释（10（-5）至10（-7））病毒贮备液（初始浓度范围为10（5）至10（8）PFU/ml）样本中提取的RNA进行成功焦磷酸测序，证明了该检测试剂盒的灵敏度。通过实时RT-PCR确定，在循环阈值范围为32至37的样本中检测到耐药性标志物。焦磷酸测序方法可以提供一个有价值的工具，用于快速检测H5 N1病毒的耐药性标志物，并有助于阐明这种变化在自然和获得性耐药性中的作用。
Resistance of influenza viruses to antiviral drugs can emerge following medication or may result from natural variation. Two classes of anti-influenza virus drugs targeting either the M2 protein (amantadine and rimantadine) or neuraminidase (NA; oseltamivir and zanamivir) are currently licensed. These drugs are expected to be important in controlling the early stages of a potential pandemic. In the present study, we describe how a pyrosequencing method can be used to rapidly detect established molecular markers of resistance to M2 blockers and NA inhibitors in influenza A (H5N1) viruses. The residues L26, V27, A30, S31, and G34 in the M2 protein were targeted for pyrosequencing. The NA residues for pyrosequencing analysis included the established markers of drug resistance (H274 and N294), as well as residues of less certain relevance (V116, I117, Q136, K150, and I222). A single pair of pyro-reverse transcription (RT)-PCR primers was designed to allow amplification of an approximately 600-nucleotide-long amplicon of the NA genes of H5N1 viruses from various clades/subclades associated with infections in humans. The sensitivity of the assay was demonstrated by the successful pyrosequencing of RNA extracted from samples of serially diluted (10(-5) to 10(-7)) virus stocks with initial concentrations ranging from 10(5) to 10(8) PFU/ml. The markers of resistance were detected in samples with threshold cycle values ranging from 32 to 37, as determined by real-time RT-PCR. The pyrosequencing approach may provide a valuable tool for rapid detection of markers of drug resistance in H5N1 viruses and facilitate the elucidation of the role of such changes in natural and acquired drug resistance.