A reliable RT-PCR/RFLP assay for the molecular classification of enterovirus reference and wild type strains to either of the two genetic clusters on the basis of 5'-UTR.

The available sequence data from a large segment of the 5'-UTR of all enterovirus reference and wild type strains were analysed in an attempt to discover possible restriction sites for reliable, serotypic identification of wild type isolates. No combination of restriction endonucleases, though, was found to produce serotype-, or group-specific haplotypes. Thirteen restriction enzymes were predicted to differentiate between representatives of the two enterovirus genetic clusters on the basis of 5'-UTR. One of these enzymes, BstOI, was tested in practice for the differentiation of 61 enterovirus reference strains from 56 different serotypes and 82 wild type strains which belong to the two genetic clusters on the basis of 5'-UTR. All the representatives of the two clusters were successfully differentiated with the specific restriction enzyme. Consequently, the simple RFLP-based assay presented here could be used as a very rapid and reliable means for the initial determination of whether a clinical isolate of unknown identity belongs to either of the 'poliovirus-like', or the 'CBV-like' genetic cluster on the basis of 5'-UTR classification of human enteroviruses.