Downloaded from www.microbiologyresearch.org by IP: 54.160.103.179 On: Fri, 27 May 2016 17:54:39 Molecular typing of enteroviruses associated with viral meningitis in Cyprus, 2000–2002 Jan Richter, Dana Koptides, Christina Tryfonos and Christina Christodoulou Correspondence Jan Richter richter@cing.ac.cy Department of Molecular Virology, Cyprus Institute of Neurology and Genetics, PO Box 23462, 1683 Nicosia, Cyprus Received 30 November 2005 Accepted 28 March 2006 Human enteroviruses are responsible for a wide spectrum of clinical diseases affecting many different organ systems. Although infection is usually asymptomatic, infections of the central nervous system manifested as meningitis or encephalitis can pose a serious public health problem, especially during outbreaks. In this study, samples from 218 patients diagnosed with enteroviral meningitis between January 2000 and December 2002 were analysed in order to assess the epidemiology of human enteroviruses as a cause of viral meningitis in Cyprus. A new typing strategy, based on partial sequencing of the 59 non-coding region (59NCR), prediction of type, and selection of type-specific primers for sensitive VP1 PCR amplification, was developed. As clustering in the 59NCR was concordant with clustering in the VP1 region, quick and reliable typing by VP1 sequencing was achieved without virus isolation in cell culture. The most frequent enterovirus serotypes identified were Human echovirus 30 (55?5 %), Human echovirus 13 (15?1 %), Human echovirus 6 (13?8 %) and Human echovirus 9 (8?3 %). Human coxsackieviruses B2, B1 and B5, Human echovirus 4, Human enterovirus 71 and Human coxsackievirus A6 represented rather rare serotypes. This is the first molecular epidemiological study of enterovirus meningitis in Cyprus. Serotype distribution corresponded basically with observations in other European countries, suggesting the spread of enteroviruses by tourism. INTRODUCTION Non-polio human enteroviruses (HEVs) belonging to the Picornaviridae family represent the most common cause of aseptic meningitis, which, conversely, is the most commonly identified illness in association with enterovirus infections (Rotbart, 1995). HEVs are ubiquitous faecal-orally trans- mitted small RNA viruses with a seasonal peak of infection in summer and autumn. Outbreaks of disease caused by a single serotype strain are frequently reported and represent a major public health problem (Ozkaya et al., 2003; Avellon et al., 2003; Thoelen et al., 2003). The typing of enteroviruses is based conventionally on virus isolation in cell culture, followed by neutralization with intersecting pools of type-specific antisera and confirmation with monospecific antisera to identify the serotype. Due to the large number of antigenically distinct serotypes, serotyp- ing procedures are time-consuming, labour-intensive and costly. Moreover, the limited supply of reference sera and their inability to detect new antigenic variants or emerging serotypes are major drawbacks of neutralization typing (el Sageyer et al., 1998). Because VP1 is the major surface-accessible protein in the picorna virion that exhibits relevant serotype-specific neu- tralization epitopes, the sequence of the VP1-encoding gene has been shown to correlate well with the classical serotype classification (Oberste et al., 1999, 2003). However, assays utilizing highly degenerated primers to amplify a portion of the VP1 gene in all HEV serotypes usually require cell culture prior to extraction due to a lack of sensitivity (Oberste et al., 1999, 2003; Caro et al., 2001). Since sample material is often used up completely for RNA and DNA extractions, for diagnostic purposes the typing of HEVs utilizing cell culture is often not possible. For this reason a typing strategy based on the sequencing of a large part of the 59 untranslated region and amplification of a part of the VP1 region was developed. In this study, samples from 218 patients with viral menin- gitis, who were diagnosed enterovirus positive between January 2000 and December 2002, have been analysed. Data obtained by sequencing a 399 bp fragment in the 59NCR of the enterovirus genome, together with informa- tion about currently circulating HEV strains, were used to Abbreviations: CSF, cerebrospinal fluid; CV, human coxsackievirus; HEV, human enterovirus; 59NCR, 59 non-coding region. The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are AY898964–AY899181. 46447 G 2006 SGM Printed in Great Britain 1035 Journal of Medical Microbiology (2006), 55, 1035–1041 DOI 10.1099/jmm.0.46447-0