Figure 3. Histogram of the observed distances (Kimura two param- eters method) within Canary Islands and Iberian Peninsula groups (dark gray) and between them (light gray). Analysis of variance (ANOVA) (one-way) test results: F=5238 (p=0.000). Distances within homologous groups: Canary Islands: mean 0.008 (standard deviation [SD] 0.004; n=351); Iberian Peninsula: mean 0.010 (SD0.006; n=666). Distances between heterologous groups (black): mean 0.033 (SD 0.005; n=999). (Postdam79), AF401357 (Berlin65), AF401356 (Chemniz00), AF401355 (Krefeld00), AF401354 (Stuttgart00), AF401353 (Ulm00), AY007223 (Weissenfels00), AF295467 (Sweden99), AF152299 (VA86-6776), AF081635 (TX95-2089), AB092985 (JA1CR0210F), AB092984 (JA1ON022R), AY131288 (KASS31001), AF538285 (DRES41001), and AF538284 (MARD37701). The sequence AF081349, corresponding to the ENV69 strain Toluca-1, was also included as the nearest possible taxon and outgroup. GenBank accession numbers of the rest of the VP1 partial gene of HEVs pro- totype strains have been detailed previously (7). Results Molecular Typing of Isolates A preliminary multiple sequence alignment, as described by Palacios et al. (11), grouped all the Spanish viruses with the EV13 reference strain with 79.5% identi- ty (SD [standard deviation] 0.37). ENV69 reference strain was the nearest taxon with 74.3% (SD 0.33) identity; the rest of the HEVs shared <71%. In addition, pairwise sequence distance comparison showed that all the isolates from Spain differed from the EV13 and ENV69 reference strains with observed nucleotide distances of 0.260 (stan- dard error [SE] 0.028) and 0.344 (SE 0.032), respectively. RESEARCH Consequently, all the Spanish isolates were classified as EV13, in agreement with the neutralization test results. Comparison of the Nucleic Acid of the Spanish Sequences Spanish isolates grouped into two major clusters corre- sponding to their respective geographic origin (Iberian Peninsula and Canary Islands) with statistical significance (Figures 2 and 3). The 10-nt changes that determined the groups were all in third codon position. The nucleotide dis- tances observed within the Canary Islands sequence group ranged from 0 to 0.022, whereas within the Iberian Peninsula group, the range was from 0 to 0.029. The dis- tance range obtained after comparison of sequences belonging to these different groups was 0.024–0.052. The study of the temporal pattern of the Spanish outbreak sug- gests that, although all the Spanish isolates have a common source, the group from the Canary Islands might be closer to the ancestor. Comparison of the Spanish Isolates with Other Available EV13 Sequences EV13 viruses isolated in 2000 and 2001 from Germany, Japan, and Spain were phylogenetically close. Within all of the studied sequences, the more similar ones, with a nucleotide distance of 0.011, corresponded to the strains isolated in Germany and the Canary Islands in 2000. The Figure 4. Phylogenetic tree of the 5' VP1 extreme showing the relationship between the Spanish isolates and others. The sequences included are the 14 Spanish echovirus 13 selected iso- lates as well as the German (n=12), Italian (n=1), Swedish (n=1), and Japanese (n=2) isolates. Phylogenetic tree reconstructed with the maximum-likelihood method, 50 Jumble. Gray sequences cor- respond to the 2001 isolates. Emerging Infectious Diseases • Vol. 9, No. 8, August 2003 937