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.
Zhou
Fei
F
Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Darcy Road, Westmead, Sydney, New South Wales 2145, Australia.
Kong
Fanrong
F
McPhie
Kenneth
K
Ratnamohan
Mala
M
Gilbert
Gwendolyn L
GL
Dwyer
Dominic E
DE
D017931:DNA Primers / Q000235:genetics; D004770:Enterovirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D004769:Enterovirus Infections / Q000821:virology*; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D011110:Polymorphism, Genetic; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D012703:Serotyping; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D015345:Oligonucleotide Probes; D012367:RNA, Viral