Molecular identification and analysis of nonserotypeable human enteroviruses

Abstract

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.

FIG. 1.

Representative RT-PCR-RLB results for 42 clinical isolates, including 40 nonserotypeable isolates and the positive control CVA9 (See Table S1 in the supplemental material for description and targets of probes listed on the left). The first membrane (shown here) of the second set of membranes was bound with 42 probes (2 HEV-specific probes and 4 newly designed serotype-specific probes [probes 5 to 8] for each of 10 common serotypes/genotypes). RT-PCR-RLB results for the isolates shown, from left to right, were CVA9 (positive control), E11, E18*, E7, E9, E30, E6*, E30, E25#, E30, E30, CVA16, E5#, E6*, E18*, E30, E9, E11, CB4*, CVA16, CVA16, CVB2, E30, E30, E9, E18*, E9, E30, E30, E9, EV71, CVA9, CVA16, E9, E30, E30, E9, E30, E9, EV71, EV71, and CVA9 (positive control). *, Isolates belonging to one of the other 10 common serotypes/genotypes were identified by serotype-specific probes on the second membrane of the second set of membranes (data not shown). #, Isolates belonging to one of the uncommon serotypes/genotypes were not identified by serotype-specific probes on both membranes but only identified by VP1 sequencing.