Identification of 20 Common Human Enterovirus Serotypes by Use of a Reverse Transcription-PCR-Based Reverse Line Blot Hybridization Assay

The more than 100 human enterovirus (HEV) serotypes can also be classified into four species, HEV-A to -D, based on phylogenetic analysis of multiple gene regions. Current molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) amplification and nucleotide sequencing of the entire or 3′ half of the VP1 gene. An RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient approach to characterize common HEVs. Twenty HEV serotypes accounted for 87.1% of all HEVs isolated at an Australian reference virology laboratory from 1979 to 2007. VP1 sequences of all known HEV prototype strains were aligned to design one sense primer and three antisense primers for RT-PCR. After sequencing of the complete VP1 genes of 37 previously serotyped examples of the commonest 20 serotypes and alignment of these VP1 sequences with GenBank sequences, four serotype-specific probes for each serotype were designed for RLB. The RT-PCR-RLB assay was then applied to 132 HEV isolates, made up of the previously sequenced 37 isolates and another 95 serotyped clinical isolates. The RT-PCR-RLB genotypes corresponded with the serotypes for 131/132 isolates; the one exception was confirmed by VP1 sequencing, and the genotype was confirmed by repeat conventional serotyping. Genotyping by RT-PCR-RLB complements traditional serotyping methods and VP1 sequencing and has the advantages of convenience, speed, and accuracy. RT-PCR-RLB allows detection of specific enteroviral serotypes or genotypes associated with HEV outbreaks and significant disease.