Assay for 5′ Noncoding Region Analysis of All Human Rhinovirus Prototype Strains
Assay for 5′ Noncoding Region Analysis of All Human Rhinovirus Prototype Strains
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DOI：
10.1128/jcm.00674-08
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发表时间：
2008-11-01
影响因子：
9.4
通讯作者：
Schnurr, David P.
中科院分区：
文献类型：
--
作者：
Kiang, David;Kalra, Ishmeet;Schnurr, David P.
越来越多的人认识到鼻病毒与严重的下呼吸道疾病之间的联系，这表明有必要了解鼻病毒的特定血清型与其临床后果之间的关系。要做到这一点，需要一种特异和灵敏的检测方法来直接从临床标本中检测和分型鼻病毒。使用培养和血清中和的传统血清分型方法非常耗时，仅限于某些参考实验室，而且由于存在100多种人类鼻病毒(HRV)血清型而变得复杂。因此，我们开发了一种基于序列的分析方法，针对约占5‘非编码区(NCR)三分之二的390bp片段。我们的目标是开发一种检测方法，允许直接从临床标本中扩增目标序列，并在所有101株鼻病毒原型株中进行区分。我们测定了该区域所有101株HRV原型毒株的序列，以区分病毒分离株和临床标本中的病毒基因型。我们在总共101个临床病毒分离株和24个临床样本中评估了这一方法，并将我们的发现与使用HRV基因组的不同区域(VP4-VP2区域)的基因分型结果进行了比较。与儿童严重呼吸道疾病相关的5份标本与任何已知的鼻病毒血清型无关，被发现属于鼻病毒的一个新的基因组组，即C组。此外，还发现了与纽约和澳大利亚发现的A2组变异以及另外两个新的A组簇(GAC1和GAC2)相对应的分离物。
Increasing recognition of the association of rhinovirus with severe lower respiratory tract illnesses has clarified the need to understand the relationship between specific serotypes of rhinovirus and their clinical consequences. To accomplish this, a specific and sensitive assay to detect and serotype rhinovirus directly from clinical specimens is needed. Traditional methods of serotyping using culture and serum neutralization are time-consuming, limited to certain reference laboratories, and complicated by the existence of over 100 serotypes of human rhinoviruses (HRVs). Accordingly, we have developed a sequence-based assay that targets a 390-bp fragment accounting for approximately two-thirds of the 5' noncoding region (NCR). Our goal was to develop an assay permitting amplification of target sequences directly from clinical specimens and distinction among all 101 prototype strains of rhinoviruses. We determined the sequences of all 101 prototype strains of HRV in this region to enable differentiation of virus genotypes in both viral isolates and clinical specimens. We evaluated this assay in a total of 101 clinical viral isolates and 24 clinical specimens and compared our findings to genotyping results using a different region of the HRV genome (the VP4-VP2 region). Five specimens associated with severe respiratory disease in children did not correlate with any known serotype of rhinovirus and were found to belong to a novel genogroup of rhinovirus, genogroup C. Isolates were also found that corresponded to the genogroup A2 variant identified in New York and Australia and two other novel group A clusters (GAC1 and GAC2).