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Journal of Clinical Microbiology, March 2004, p. 963-971, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.963-971.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Analysis of the Serotype and Genotype Correlation of VP1 and the 5' Noncoding Region in an Epidemiological Survey of the Human Enterovirus B Species

Laboratory of Clinical and Epidemiological Virology, Department of Microbiology and Immunology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium,¹ Department of Chemistry and Biomedical Sciences, University of Kalmar, Kalmar, Sweden²

Received 28 August 2003/ Returned for modification 27 October 2003/ Accepted 28 November 2003

The sequence identity of the enterovirus VP1 gene has been shown to correlate with the serotype concept. Enterovirus molecular typing methods are therefore often based on sequencing of the VP1 genomic region and monophyletic clustering of VP1 sequences of a homologous serotype. For epidemiological surveillance, 342 enterovirus samples obtained from patients with aseptic meningitis in Belgium from 1999 to 2002 were first diagnosed as being enterovirus positive by amplification of the 5' noncoding region (5'NCR) by reverse transcription (RT)-PCR. Subsequently, samples were molecularly typed by RT-nested PCR amplification and sequencing of a portion of the VP1 gene. Phylogenetic analyses were performed to investigate enteroviral evolution and to examine the serotype and genotype correlation of the two genomic regions. Our typing results demonstrated echovirus 30, echovirus 13, echovirus 18, and echovirus 6 to be the most predominant types. Echoviruses 13 and 18 were considered to be emerging human serotypes since 2000 and 2001, respectively, as they had been rarely reported before. Several serotypes existed as multiple genotypes (subtypes) from 1999 to 2002, but genomic differences mainly resided at synonymous sites; these results strongly suggest that the subtypes exhibit similar antigenic properties. Phylogenetic analyses confirmed that VP1 is an adequate region for molecular typing. Serotype-specific clusters are not observed commonly in phylogenetic trees based on the 5'NCR, and the phylogenetic signal in the 5'NCR was found to be particularly low. However, some substructure in the 5'NCR tree made a tentative prediction of the enterovirus type possible and was therefore helpful in PCR strategies for VP1 (e.g., primer choice), provided some background knowledge on the local spectrum of enteroviruses already exists.

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