RESEARCH study of the temporal pattern by the maximum-likelihood approach also grouped the German 2000 isolates with the Spanish ones (Figure 4). For the European strains, obtained before 2000, the iso- lates were genetically more distant from the Spanish iso- lates; the 1999 strain from Sweden was the second most similar to the Spanish isolates, followed by the isolate from Italy (1996) and those from Germany (1979, 1976, and 1965). The two strains from the United States (VA86- 6776 and TX95-2089) were the most distant from the Spanish isolates, with a genetic distance of 0.345 and 0.311, respectively (Figure 2). These two viruses seemed to be as similar to ENV69 as to EV13. Further sequence analysis of other strains of American EV13 and additional ENV69 isolates could clarify this point. However, although no other ENV69 sequences were available for comparison, we could confirm that these sequences did not cluster into the same evolutionary lineage of all other pub- lished EV13. Comparison of Amino Acid Sequences Several amino acid differences could be observed between the Spanish isolates and the EV13 reference strain. The EV13 neutralization sites have not been deter- mined. However, they have been reported for other ENVs and some of them (sites I and III) are in the same position and have the same amino acids involved. For this reason, the amino acid changes noticed in this study and neutral- ization sites previously reported for other ENVs were pro- jected on the three-dimensional EV11 structure (Figure 5). The possible relationship between them was detailed; however, we can only speculate about whether EV13 neu- tralization sites were affected or not. Two changes, A100V and F238I, were structurally close to known neutralization site I described for poliovirus (13), CBV4 (32), and CBV5 and SVDV (14). However, in most cases the amino acid differences were located in the VP1-2A junction (S274A, T276P, N284S, P285T, A286G, S287G, K288R, M290V, N291T, and H292N), affecting a region that was consid- ered part of the neutralizing antigenic site III for SVDV (15). The amino acid changes (S274A and T276P) implied a change in the properties from hydrophilic to hydrophobic residues. These two positions were also different from the reference strain in the two U.S. isolates; however, they did not result in amino acids with hydrophobic characteristics (T276D/E). Apart from the American isolates, the comparison of the amino acid sequence for EV13 isolates with respect to the reference strain was only possible in the amino termi- nal part of the VP1 protein. In this region, changes were identified in the strains that dated from 1965 to 1979, mostly affecting amino acids that have been considered as part of the neutralizing antigenic site I in poliovirus (D84S and A85D/E) (13). Only one amino acid (M216I) differed between the two Spanish clusters. This position was not related to any known antigenic site. Discussion After a 12-years period in which EV13 circulation in Spain was not detected, a virus identified through neutral- ization test as EV13 began to be isolated in CSF of patients with aseptic meningitis. These isolates were also clearly identified as EV13 serotypes through molecular typing by their substantial homology with the EV13 Del Carmen Figure 5. Mapping of the echovirus (EV) 13 VP1 amino acids. The three-dimensional structure is projected onto EV11 structure (GenBank accession no. 1H8TA) with VP1 amino acid numbering according to EV11. The sequenced fragment is shown in red. Previously reported neutralizing antigenic sites for poliovirus, CBV4, and SVDV are shown in yellow. The enterovirus canyon structure is depicted in a similar location to the canyon structure of poliovirus as reported by (40). 938 Emerging Infectious Diseases • Vol. 9, No. 8, August 2003