Mirand Audrey, Archimbaud Christine, Henquell Cécile, Michel Yanne, Chambon Martine, Peigue-Lafeuille Hélène, Bailly Jean-Luc
CHU Clermont-Ferrand, Laboratoire de Virologie, Centre de Biologie, Clermont-Ferrand, France.
J Med Virol. 2006 Dec;78(12):1624-34. doi: 10.1002/jmv.20747.
Enteroviruses (EVs) represent the main etiological agents of epidemics of viral meningitis and especially the serotypes related to the human enterovirus B species. Genetic typing by sequencing a PCR-amplified portion of the genome has proved to be useful for identifying EVs and is more rapid than standard seroneutralization tests. However, prospective genotyping has not been reported in routine practice within a clinical diagnostic laboratory. A genetic typing assay using two sets of primers was developed for the amplification and sequencing of the VP1 coding sequence of the HEV-B serotypes. Identification was carried out by sequence comparisons with EV sequences in GenBank using the BLAST search tool and confirmed by phylogenetic analysis. This method was used to identify prospectively the 48 enteroviruses isolated in patients with either enterovirus-proved meningitis (n = 41) or other clinical manifestations (n = 7) admitted to the University Hospital of Clermont-Ferrand (France) in 2005. The assay was also used to type retrospectively EVs isolated in cerebrospinal fluid specimens of 25 patients admitted to the Trousseau Paediatric Hospital in Paris (France) between February and August 2005. In both prospective and retrospective investigations of meningitis, echovirus 30 (E30) was the most frequent serotype, followed in decreasing order by E18, E13, coxsackievirus B5, B3, E6, E4, E7, E11, E33, and coxsackievirus A9. In patients with other manifestations, coxsackievirus B3, B5, and E3 were each identified twice, and E2 once. In E30 infected patients, nine different lineages were demonstrated by phylogenetic analysis. Genetic typing allowed the prospective, effective and rapid identification of all EV isolates involved in the 2005 outbreak. Molecular typing in combination with phylogenetic analysis will be a reliable means to confirm the emergence of new EV variants, and is of interest of both individual patients and public health.
肠道病毒（EVs）是病毒性脑膜炎流行的主要病原体，尤其是与人类肠道病毒B种相关的血清型。通过对基因组的PCR扩增部分进行测序进行基因分型已被证明有助于鉴定肠道病毒，并且比标准血清中和试验更快。然而，临床诊断实验室的常规实践中尚未报告前瞻性基因分型。开发了一种使用两组引物的基因分型测定法，用于扩增和测序HEV-B血清型的VP1编码序列。通过使用BLAST搜索工具与GenBank中的EV序列进行序列比较来进行鉴定，并通过系统发育分析进行确认。该方法用于前瞻性鉴定2005年在法国克莱蒙费朗大学医院收治的经肠道病毒证实为脑膜炎的患者（n = 41）或其他临床表现患者（n = 7）中分离出的48株肠道病毒。该测定法还用于回顾性分析2005年2月至8月期间在法国巴黎特鲁索儿童医院收治的25例患者脑脊液标本中分离出的肠道病毒。在脑膜炎的前瞻性和回顾性调查中，埃可病毒30（E30）是最常见的血清型，其次依次为E18、E13、柯萨奇病毒B5、B3、E6、E4、E7、E11、E33和柯萨奇病毒A9。在有其他表现的患者中，柯萨奇病毒B3、B5和E3各被鉴定出2次，E2被鉴定出1次。在E30感染患者中，系统发育分析显示出9种不同的谱系。基因分型使得能够前瞻性、有效且快速地鉴定出参与2005年疫情爆发的所有肠道病毒分离株。分子分型与系统发育分析相结合将是确认新的肠道病毒变体出现的可靠手段，对个体患者和公共卫生都具有重要意义。
