Molecular Identification and Analysis of Nonserotypeable Human Enteroviruses

Conventional approaches to characterizing human enteroviruses (HEVs) are based on viral isolation and neutralization. Molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) and nucleotide sequencing of the entire or partial VP1 gene. A modified RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient way to characterize common and nonserotypeable (by neutralization) HEVs. Twenty HEV serotypes accounted for 87.1% of all HEVs isolated at a reference laboratory from 1979 to 2007; these common serotypes were identified using one sense and three antisense primers and a set of 80 serotype-specific probes in VP1 (F. Zhou et al., J. Clin. Microbiol. 47: 2737-2743, 2009). In this study, one HEV-specific primer pair, two probes in the 5' untranslated region (UTR), and a new set of 80 serotype-specific probes in VP1 were designed. First, we successfully applied the modified RT-PCR-RLB (using two HEV-specific probes and two sets of serotype-specific probes) to synchronously detect the 5' UTR and VP1 regions of 131/132 isolates previously studied (F. Zhou et al., J. Clin. Microbiol. 47: 2737-2743, 2009). Then, this method was used to identify 73/92 nonserotypeable HEV isolates; 19 nonserotypeable isolates were hybridized only with HEV-specific probes. The VP1 region of 92 nonserotypeable HEV isolates was sequenced; 73 sequences corresponded with one or both RLB results and 19 (not belonging to the 20 most common genotypes) were identified only by sequencing. Two sets of serotype-specific probes can capture the majority of strains belonging to the 20 most common serotypes/genotypes simultaneously or complementarily. Synchronous detection of the 5' UTR and VP1 region by RT-PCR-RLB will facilitate the identification of HEVs, especially nonserotypeable isolates.