Genetic diversity among 107 coxsackievirus B4 field isolates has been studied. These isolates included clinical and environmental isolates originating from Finland, the Netherlands and France, and also from several other countries, including the USA. Three genomic regions were used for phylogenetic analyses: the VP1/2A junction, the entire VP1 and the VP4/VP2 region. Alignment of the deduced amino acid sequence in the VP1/2A junction revealed extensive sequence variation at the previously proposed cleavage site. MS analysis of proteolytic fragments from VP1 revealed that the exact cleavage site is situated between amino acid residues Thr-849 and Gly-850. At least seven distinct genetic lineages, or genotypes, had been circulating in Europe during the period 1959-1998. Two genotypes were endemic in the Netherlands during most of the investigated period. Genetically closely related strains could be found in different countries, and different genotypes co-circulated at the same time in a given country. Clustering patterns were identical in the three genomic intervals. In the VP4/VP2 region, the intraserotypic variation approached interserotype variation. Sequence comparisons of the entire VP1 gene gave a reliable genetic identification of enterovirus serotype. It is suggested that, for genotype classification of previously serotyped coxsackievirus B4 isolates, comparison of VP1/2A sequences is sufficient, but for more detailed investigation of genetic relationships, and for 'genetic serotyping', the entire VP1 gene should be used. The VP4/VP2 region is less reliable for genetic serotyping and genotyping, although the primers are able to amplify many different serotypes.
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