Molecular strategy for 'serotyping' of human enteroviruses

To explore further the phylogenetic relationships between human enterovirus es and to develop new diagnostic approaches, we designed a pair of generic primers in order to study a 1452 bp genomic fragment (relative to the polio virus Mahoney genome), including the 3' end of the VP1-coding region, the 2 A- and 2B-coding regions, and the 5' moiety of the 2C-coding region. Fifty- nine of the 64 prototype strains and 45 field isolates of various origins, involving 21 serotypes and 6 strains untypeable by standard immunological t echniques, were successfully amplified with these primers. By determining t he nucleotide sequence of the genomic fragment encoding the C-terminal thir d of the VP1 capsid protein we developed a molecular typing method based on RT-PCR and sequencing. If field isolate sequences were compared to human e nterovirus VP1 sequences available in databases, nucleotide identity score was, in each case, highest with the homotypic prototype (74.8 to 89.4%). Ph ylogenetic trees were generated from alignments of partial VP1 sequences wi th several phylogeny algorithms. In all cases, the new classification of en teroviruses into five identified species was confirmed and strains of the s ame serotype were always monophyletic. Analysis of the results confirmed th at the 3' third of the VP1-coding sequence contains serotype-specific infor mation and can be used as the basis of an effective and rapid molecular typ ing method. Furthermore, the amplification of such a long genomic fragment, including non-structural regions, is straightforward and could be used to investigate genome variability and to identify recombination breakpoints or specific attributes of pathogenicity.