RESEARCH totype strains (Del Carmen and Toluca-1, respectively), and the isolates VA86-6776 (30) and TX95-2089 (7). For the analysis of the 279-nt VP1 5′end fragment (nt 2609–2887, according to the EV11 polyprotein genome, X80059), sequences available from GenBank of the fol- lowing isolates were included: 12 isolates from Germany, obtained during 1965, 1974, 1976, 1979, 2000, and 2001; one isolate each from Italy (1996) and Sweden (1999), and two from Japan (2001). This second group of sequences was compared with the corresponding VP1 5′ end gene of 14 Spanish representative isolates selected according to their geographic origin (four from the Canary Islands and within the Iberian Peninsula, four from the north, four from the center of the peninsula, and two from the east). Multiple sequence alignments were built with Clustal X. For the molecular typing, Spanish VP1 3′ end gene sequences were compared pairwise with the HEV proto- type strains to obtain the identity score (11). The percent- age of similarity of the isolates with respect to the refer- ence strain was calculated through Megalign software (DNASTAR, Madison, WI). The phylogenetic tree in Figure 2 was reconstructed through the neighbor-joining method (MEGA version 2.1 software package; available from URL: http://www.mega- software.net) by using Kimura two parameters as substitu- tion model, with statistical significance of phylogenies estimated by bootstrap analysis with 1,000 pseudoreplicate datasets. The transition-transversion ratio employed (TRatio=2) was estimated through the Tree-puzzle version 5.0 software (available from: URL: http://www.tree-puz- zle.de). Genetic distances were calculated using the same model of nucleotide substitution. The distance matrix was recorded and the pairwise observed distance values were employed to define different subgroups and to build the histogram in Figure 3. The analysis of variance one-way test, with a statistical significance value of 0.05, was used to compare the means of the pairwise observed distance value matrix. To reconstruct the phylogenetic tree shown in Figure 4, the maximum-likelihood method (DNAML program, PHYLIP software [available from: URL: http://evolution.genetics.washington.edu]), was used, with hidden Markov as the model of nt substitution. A Poisson correction model was used to compare the amino acid sequences. Three-Dimensional Structure The VP1 amino acid changes of the Spanish isolates with respect to the EV13 reference strain were projected onto the three-dimensional structure of EV11 (GenBank 1H8TA) (31) by using the program Cn3D (available from: URL: http://www.ncbi.nlm.nih.gov/Structure). Neutral- izing antigenic sites previously reported for poliovirus (13), CBV4 (32), and CBV5 and SVDV (14,15) were 936 Emerging Infectious Diseases • Vol. 9, No. 8, August 2003 localized in the structure. Amino acids were numbered according to EV11 sequence. Nucleotide Sequence Accession Numbers Fourteen complete gene sequences of Spanish EV13 VP1 were submitted to the GenBank database under accession numbers AY227334–AY227347. Fifty Spanish EV13 VP1 3′ end fragments were also submitted under accession numbers AY227284–AY227333. These EV13 sequences were also included in this study: AF081327 (Del Carmen), AJ309256 (Roma96), AF401360 (Halle74), AF401359 (Postdam76), AF401358 Figure 2. Phylogenetic tree of the VP1 3' terminal region, which identifies the Spanish isolates as echovirus 13 (EV13) and differ- entiates two clusters (Iberian Peninsula and Canary Islands). The sequences included are, apart from the reference strains, all the Spanish EV13 (61 isolates and 3 sequences obtained directly from cerebrospinal fluid) and two American isolates. Model of nucleotide substitution: Kimura two parameters. Phylogenetic tree reconstructed with the neighbor joining method, and bootstrap analysis with 1,000 pseudoreplicate datasets.