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 enteroviruses directly from clinical samples. These assays amplified short fragments from different genomic regions codifying for three proteins: VP2, VP1, and RNA polymerase. Given that enteroviruses have a high rate of degeneration within target codons among serotypes, the primers used consisted of mixed base and deoxyinosine residues. These techniques detected at 0.03-0.003 TCID50 of prototype Poliovirus 1 and Echovirus 30. They were used to characterize the enteroviral RNA detected in 18 CSF, stool, and throw swab samples and in 8 enterovirus isolates from patients with several syndromes. Phylogenetic analysis in each independent sequenced region grouped the enterovirus into four clusters, enabling genetic classification. A comparative study 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 independently in the three regions showed the highest yield of correlation between nucleotide sequences of all prototype serotypes and the analyzed genotypes 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 these were available (20/21, 95% Clustal; 12/21, 57% M-NW). These assays were useful for molecular identification of enterovirus directly from samples even when isolation was not possible.
Casas
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I
Service of Diagnostic Microbiology, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain. icasas@isciii.es
Palacios
G F
GF
Trallero
G
G
Cisterna
D
D
Freire
M C
MC
Tenorio
A
A
D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D002648:Child; D002675:Child, Preschool; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004770:Enterovirus / Q000145:classification* / Q000201:enzymology / Q000235:genetics; D004769:Enterovirus Infections / Q000821:virology*; D006801:Humans; D007223:Infant; D008969:Molecular Sequence Data; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D036022:Capsid Proteins; C442554:VP2 protein, Echovirus; C442541:VP2 protein, Poliovirus; D012321:DNA-Directed RNA Polymerases