Thoelen Inge, Lemey Philippe, Van Der Donck Ingrid, Beuselinck Kurt, Lindberg A Michael, Van Ranst Marc
Laboratory of Clinical and Epidemiological Virology, Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Belgium.
J Med Virol. 2003 Jul;70(3):420-9. doi: 10.1002/jmv.10412.
Non-polio enteroviruses are the most common cause of aseptic meningitis worldwide. From May to September 2000, a major outbreak of aseptic meningitis occurred in Belgium. Cerebrospinal fluid samples (CSF) of 122 patients were found to contain enterovirus RNA using diagnostic RT-PCR that targeted a 231-bp gene fragment in the 5' noncoding region. In addition, a molecular typing method was developed based on RT-nested PCR and sequencing directly from CSF(a) 358-bp fragment in the aminoterminal part of the VP1 capsid protein. To identify the enterovirus type, nucleotide sequences of the VP1 amplicons were compared to all the enterovirus VP1 sequences available in GenBank. Echovirus 30 (31.2%), echovirus 13 (23.8%), and echovirus 6 (20.5%) were identified most frequently during the epidemic. Coxsackievirus B5 was present in 15.6% of the samples, and could be subdivided in two distinct epidemic clusters, coxsackievirus B5a (10.7%) and B5b (4.9%). Other enteroviruses encountered were echovirus 16 (5.7%), echovirus 18 (1.6%), coxsackievirus B4 (0.8%) and echovirus 7 (0.8%). The high prevalence of echovirus 13, considered previously a rare serotype, indicates it is an emerging epidemic type. To verify the typing results and to explore further the intratypical genetic variation, phylogenetic analysis was carried out. Geographical clustering of most of the strains within each type and subtype could be observed. The RT-nested PCR strategy, carried out directly on clinical samples, is a simple and rapid method for adequate molecular typing of the Group B enteroviruses causing aseptic meningitis.
非脊髓灰质炎肠道病毒是全球无菌性脑膜炎最常见的病因。2000年5月至9月，比利时发生了一次无菌性脑膜炎大暴发。使用针对5'非编码区231bp基因片段的诊断性逆转录聚合酶链反应（RT-PCR），在122例患者的脑脊液样本（CSF）中发现含有肠道病毒RNA。此外，基于RT巢式PCR和直接从脑脊液中测序开发了一种分子分型方法：（a）VP1衣壳蛋白氨基末端部分的358bp片段。为鉴定肠道病毒类型，将VP1扩增子的核苷酸序列与GenBank中所有可用的肠道病毒VP1序列进行比较。在此次流行期间，最常鉴定出的是埃可病毒30型（31.2%）、埃可病毒13型（23.8%）和埃可病毒6型（20.5%）。柯萨奇病毒B5存在于15.6%的样本中，可细分为两个不同的流行簇，柯萨奇病毒B5a（10.7%）和B5b（4.9%）。其他检出的肠道病毒有埃可病毒16型（5.7%）、埃可病毒18型（1.6%）、柯萨奇病毒B4型（0.8%）和埃可病毒7型（0.8%）。埃可病毒13型以前被认为是一种罕见血清型，其高流行率表明它是一种新兴的流行类型。为验证分型结果并进一步探索型内遗传变异，进行了系统发育分析。可观察到每种型和亚型内大多数毒株的地理聚类情况。直接在临床样本上进行的RT巢式PCR策略是对引起无菌性脑膜炎的B组肠道病毒进行充分分子分型的一种简单快速的方法。
