Incorporation of a Proteotyping Approach Using Mass Spectrometry for Surveillance of Influenza Virus in Cell-Cultured Strains
Incorporation of a Proteotyping Approach Using Mass Spectrometry for Surveillance of Influenza Virus in Cell-Cultured Strains
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DOI：
10.1128/jcm.02315-13
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发表时间：
2014-03-01
影响因子：
9.4
通讯作者：
Downard, Kevin M.
中科院分区：
文献类型：
--
作者：
Fernandes, Neil D.;Downard, Kevin M.
致命的大流行性流感病毒株的重新出现使得有必要开发用于流感病毒的快速检测和分型的改进方法，这将使得能够在分子水平上表征更多的毒株。将来自主要临床标本的人流感病毒的代表性循环毒株在细胞培养物中生长，通过聚乙二醇沉淀纯化，用内切蛋白酶进行蛋白水解消化，并使用A型H1 N1和H3 N2以及B型流感病毒特征性的独特特征肽通过高分辨率质谱法进行分析和鉴定。这种蛋白质分型方法能够对A型流感病毒的循环毒株进行分型和亚型，区分共同循环的季节性和大流行性H1N1病毒，并通过高分辨率质谱的单离子检测确定B型病毒的谱系。使用与在细胞培养物中生长的临床标本的逆转录（RT）-PCR测定中使用的病毒滴度相当的病毒滴度获得结果。该方法是一种比RT-PCR更快速和直接的方法，可以整合到目前用于监测新出现的大流行性和季节性流感病毒的现有程序中。
The reemergence of deadly pandemic influenza virus strains has necessitated the development of improved methods for rapid detection and subtyping of influenza viruses that will enable more strains to be characterized at the molecular level. Representative circulating strains of human influenza viruses from primary clinical specimens were grown in cell culture, purified through polyethylene glycol precipitation, proteolytically digested with an endoproteinase, and analyzed and identified by high-resolution mass spectrometry using unique signature peptides that are characteristic of type A H1N1 and H3N2 and type B influenza viruses. This proteotyping approach enabled circulating strains of type A influenza virus to be typed and subtyped, cocirculating seasonal and pandemic H1N1 viruses to be differentiated, and the lineage of type B viruses to be determined through single-ion detection by high-resolution mass spectrometry. Results were obtained using virus titers comparable to those used in reverse transcription (RT)-PCR assays with clinical specimens grown in cell cultures. The methodology represents a more rapid and direct approach than RT-PCR and can be integrated into existing procedures currently used for the surveillance of emerging pandemic and seasonal influenza viruses.