Molecular characterization of human enteroviruses in clinical samples: Comparison between VP2, VP1, and RNA polymerase regions using RT nested PCR assays and direct sequencing of products

Three nested RT-PCR assays were developed to permit sensitive typing of ent eroviruses directly from clinical samples. These assays amplified short fra gments from different genomic regions codifying for three proteins: VP2, VP 1, and RNA polymerase. Given that enteroviruses have a high rate of degener ation within target codons among serotypes, the primers used consisted of m ixed base and deoxyinosine residues. These techniques detected at 0.03-0.00 3 TCID50 of prototype Poliovirus 1 and Echovirus 30. They were used to char acterize the enteroviral RNA detected in 18 CSF, stool, and throw swab samp les and in 8 enterovirus isolates from patients with several syndromes. Phy logenetic analysis in each independent sequenced region grouped the enterov irus into four clusters, enabling genetic classification. A comparative stu dy was performed among the 26 sequences obtained after direct sequencing of products with those available in the nucleotide databases. The efficiency of each assay for enterovirus identification was evaluated by both distance (Clustal) and similarity(M-NW) indices. Comparative results obtained indep endently in the three regions showed the highest yield of correlation betwe en nucleotide sequences of all prototype serotypes and the analyzed genotyp es in the VP1 region (26/26, 100% Clustal; 22/26,85% M-NW). Conversely, the VP2 region failed to identify some of the circulating enteroviruses (17/26 ,65% Clustal; 16/26, 62% M-NW). Using the RNA polymerase region, sequences from samples and isolates were associated with prototype strains whenever t hese were available (20/21, 95% Clustal; 12/21, 57% M-NW). These assays wer e useful for molecular identification of enterovirus directly from samples even when isolation was not possible. (C) 2001 Wiley-Liss, Inc.