Direct broad-range detection of alphaviruses in mosquito extracts
Direct broad-range detection of alphaviruses in mosquito extracts
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DOI：
10.1016/j.virol.2007.06.016
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发表时间：
2007-11-25
期刊：
影响因子：
3.7
通讯作者：
Ecker, David J.
中科院分区：
文献类型：
--
作者：
Eshoo, Mark W.;Whitehouse, Chris A.;Ecker, David J.
甲病毒属的成员是一组主要由蚊子传播的RNA病毒。甲病毒至少有29种和更多的亚型，其中一些被认为是潜在的生物武器。我们开发了一种多位点RT-PCR后电喷雾电离质谱（RT-PCR/ESI-MS）检测方法，该方法使用扩增子碱基组成来检测和鉴定甲病毒。一小组靶向甲病毒RNA基因组保守位点的引物对用于扩增一组36个病毒分离物，这些分离物代表了特征的旧大陆和新世界甲病毒。扩增结果的碱基组成可明确地确定36株菌株中的35株的种或亚型。该检测在没有培养的情况下，在由实验室感染和未感染蚊子组成的池中检测到委内瑞拉马脑炎病毒（VEEV）、东部马脑炎病毒（EEEV）以及两者的混合物。此外，该方法还用于检测从南美和亚洲收集的自然存在的蚊虫媒介中的甲病毒。在秘鲁伊基托斯附近收集的蚊子池中发现含有一种具有非常明显特征的甲病毒。随后的序列分析证实该病毒是Mucambo病毒种（VEEV复合体中的HID亚型）的成员。我们开发的检测方法为甲型病毒的监测提供了快速、准确和高通量的检测方法。(c) 2007爱思唯尔公司版权所有。
Members of the genus Alphavirus are a diverse group of principally mosquito-borne RNA viruses. There are at least 29 species and many more subtypes of alphaviruses and some are considered potential bioweapons. We have developed a multi-locus RT-PCR followed by electrospray ionization mass spectrometry (RT-PCR/ESI-MS) assay that uses the amplicon base compositions to detect and identify alphaviruses. A small set of primer pairs targeting conserved sites in the alphavirus RNA genome were used to amplify a panel of 3 6 virus isolates representing characterized Old World and New World alphaviruses. Base compositions from the resulting amplicons could be used to unambiguously determine the species or subtype of 35 of the 36 isolates. The assay detected, without culture, Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), and mixtures of both in pools consisting of laboratory-infected and -uninfected mosquitoes. Further, the assay was used to detect alphaviruses in naturally occurring mosquito vectors collected from locations in South America and Asia. Mosquito pools collected near Iquitos, Peru, were found to contain an alphavirus with a very distinct signature. Subsequent sequence analysis confirmed that the virus was a member of the Mucambo virus species (subtype HID in the VEEV complex). The assay we have developed provides a rapid, accurate, and high-throughput assay for surveillance of alphaviruses. (c) 2007 Elsevier Inc. All rights reserved.