Molecular typing and epidemiology of enteroviruses identified from an outbreak of aseptic meningitis in Belgium during the summer of 2000

Abstract

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.