Molecular identification of enterovirus by analyzing a partial VP1 genomic region with different methods

Abstract

VP1 is the most suitable region for use in the identification of enterovirus. Although VP1 sequencing methods may vary, it is necessary to agree on a common strategy of sequence analysis. Identification of a strain type may be achieved by three different approaches: pairwise sequence alignment, multiple-sequence alignment, and phylogenetic inference. Other methods are also available, but they are not simple enough to be performed at a virology laboratory. The performances of these methods were evaluated with nucleotide and protein sequences obtained from 32 original samples, 8 enterovirus isolates, and 64 GenBank sequences. Pairwise sequence alignment methods had very different results. The DNASTAR package identified only 28.8% of enterovirus strains, while the Genetics Computer Group package identified 50.0 or 72.1% of enterovirus strains when nucleotide or amino acid sequences were analyzed, respectively. Multiple-sequence alignment methods identified 94.2% (Clustal W program) or 92.3% (Pileup program) of the enterovirus strains, while the phylogenetic method increased this rate to 99.0%. Comparative evaluation of these analysis methods showed that the Clustal W program (version 1.81), a freely available multiple-sequence alignment program, presented one of the best performances when used with the correct criteria. Other commercial and expensive programs did not achieve the same performances, making them less suitable for molecular typing of enteroviruses. Finally, although phylogenetic inference is the most demanding method in terms of knowledge of the user, it remained the best option analyzed.

FIG. 1.

Results from each test comparing the performances of the programs. The program tested the combinations of GEP and GOP parameters shown for each indicator with each alignment test. The program and the GEP parameters used are shown in the rows. Columns show the GOP parameter for each alignment. The box to the right of each comparison describes the symbols used for the scores. (a) Column score result for deduced amino acid alignments. (b) Percent overall similarity result for each amino acid alignment. (c) Column score result for nucleotide alignments. (d) Percent overall similarity result for each nucleotide alignment. (e) Variation in overall similarity of the alignment considering different delay divergent values with the Clustal W program. The GOP and GEP scores were set equal to 100. The default delay divergent value was increased by 5 units up to a value of 95.

FIG. 2.

Consensus phylogenetic tree constructed with the PHYLIP package for 104 EV sequences. Alignments were obtained with the Clustal W program. The statistical significance of the phylogenies constructed was estimated with the SEQBOOT program. The 100 pseudoreplicate data sets obtained were analyzed with the DNADIST program with the parameters of the Kimura 2 model of nucleotide substitution. The observed nucleotide distance matrix was then processed with the KITSCH program, and the tree generated was treated with the CONSENSE program. The tree was displayed with the TREEVIEW program. The numbers at the nodes represent the percentage of 100 bootstrap pseudoreplicates that contained the cluster distal to the node.