IDENTIFICATION OF VACCINE-RELATED POLIOVIRUSES BY HYBRIDIZATION WITH SPECIFIC RNA PROBES

We developed RNA probes for the identification of poliovirus isolates by blot hybridization. Two sets of vaccine strain-specific probes were prepared. They complemented variable genomic domains within (i) the 5 '-untranslated region and (ii) the amino-terminal codons of VP1. An en terovirus group probe (EV/5UT) matching highly conserved 5'-untranslat ed region sequences was used to estimate the quantities of poliovirus (or enterovirus) RNA in the samples. Poliovirus sequences amplified fr om Sabin strain virion RNA templates by PCR were inserted into the pUC 18 plasmid vector. The antisense PCR primer for each probe set contain ed sequences encoding a T7 promoter. Hybrids were detected by a sensit ive nonisotopic method. RNA probes were labeled by incorporation of di goxigenin-uridylate into the transcripts. The binding of probe to immo bilized poliovirus RNAs was visualized by hydrolysis of the chemilumin escent substrate 4-methoxy-4-(3-phosphate-phenyl) -spiro-(1,2-dioxetan e-3,2 '-adamantane) catalyzed by alkaline phosphatase conjugated to an ti-digoxigenin (Fab) fragments. The specificities of the probes were e valuated with a panel of poliovirus isolates that had previously been characterized by sequence analysis. The RNAs of vaccine-related isolat es hybridized with the appropriate probe sets. Wild polioviruses repre senting a broad spectrum of contemporary genotypes were recognized by the inabilities of their genomes to form stable hybrids with the Sabin strain-specific probes.