prototype strain. The viruses could also be identified directly from most of the CSF samples assayed (three of them included in this study; other data not shown) without the need of isolation, demonstrating the utility and reliabil- ity of the ENV molecular typing according to the VP1 homology with respect to the reference strains (11). Indeed, the molecular study was successfully completed, despite the high degree of genetic differences between the EV13 recent isolates and the prototype strains of EV13 and ENV69, which were isolated in the Philippine Islands in 1953 and in Mexico before 1973, respectively. Phylogenetic analysis of the nucleotide sequences showed that the Spanish strains were clustered into two distinct groups, those from the Canary Islands and those from the Iberian Peninsula, coinciding with their geo- graphic origin. The first aseptic meningitis case caused by EV13 was described in the Canary Islands in February 2000. The distinctness of the groups was indicated by sev- eral silent nucleotide changes and one amino acid change, which apparently developed in a short period (the initial isolate of both groups was obtained about 1 month apart); for this reason, we cannot rule out the possibility that both groups could have had a common form but different ances- tor. The special climate of the Canary Islands could explain the winter appearance of the cases (first case February 14). When both Spanish clusters were genetically compared with isolates from other geographic origins, the EV13 strains from the Canary Islands appeared more closely related to the German isolates of 2000. This may reflect the introduction of EV13 from the Canary Islands into north- ern Europe (or vice versa) through tourist travel. The European isolates (Swedish, Italian, and German) obtained before 2000 presented nucleotide distances in respect to the Spanish isolates that increased over time. On the other hand, the two American strains, obtained in 1986 and 1995, showed the most distance between isolates. Indeed, the Spanish isolates showed larger differences with respect to the American isolates than those identified with the rest of the available EV13 sequences. In addition, the American strains of EV13 seem to be unrelated to the EV13 strains reported in the rest of the world; these American strains may represent another evolutionary line- age of this serotype. As a result, European and American EV13 strains could be assigned to different subgroups, in contrast to EV30 strains, which do not seem to be restrict- ed geographically (12). However, more U.S. isolates from the same period would be necessary to define a consistent subgroup. Yet, preliminary data show that the reported U.S. isolates from 2001 may be genetically close to the European isolates (33). The correlation between the genetic distance and the time parallels the behavior of such other epidemic viruses as EV30 (12), EV7 (34), EV25 (5), and ENV71 (35). RESEARCH However, these viruses usually maintain their isolation rates and increase their circulation periodically (28). In contrast, EV13 was isolated infrequently throughout the world until 2000, when isolation rates increased dramati- cally in several countries in association with aseptic meningitis outbreaks. This type of presentation was previ- ously described in other HEVs such as EV4, which caused a large aseptic meningitis outbreak in 1997 in Israel (36). This behavior may be due to a genetic and antigenic drift that changes the virologic properties of the isolates. However, EV13 may simply be rare, and artifacts of sur- veillance or identification may have led to its seemingly sudden appearance. In consequence, viruses could have increased in virulence or improved their pathogenic mech- anisms by antigenic change, despite the fact that they were able to be neutralized in vitro with horse-specific serum. However, further study is needed to investigate their anti- genic characteristics, the human immune response to EV13, and whether a change in virulence occurs. The study of the VP1 genome region permitted us to observe the characteristics of the major capsid protein, which may be involved in this type of process, together with the rest of structural proteins. However, other genome changes, including those affecting replication (37,38), may also be implicated; thus, to understand the molecular epidemiolo- gy of HEV outbreaks, molecular studies of other regions of the genome are indicated. Changes in EV13 coding sequence involved several differences in the C-terminal extreme of the VP1 protein, known to be highly variable and containing neutralizing antigenic site III in SVDV (15). Similar alterations have been related to positive selection affecting other ENV as EV30 (12), determining its evolution over the time. These amino acid changes in EV13 VP1 protein could be involved in the notable increase in the number of isolations of these viruses, since they were able to cause aseptic meningitis outbreaks worldwide (19–23,25,27,39). Moreover, although the isolates were successfully neutral- ized in vitro with the Lim-Benyesh-Melnick immune serum pools, the amino acid changes affecting VP1 should be taken into account during the evaluation of the neutral- ization test results. The only amino acid change between the two mentioned Spanish groups (M216I), which entails a change of a hydrophilic amino acid into a hydrophobic one, might confer some biologic advantage. It is not relat- ed to any neutralizing antigenic site previously described for ENVs, but might be related to the canyon structure, since this position is located in the floor of this structure in polioviruses (40). However, further research on the inter- action between the cellular receptor and EV13 canyon should address this point. The canyon structure is a capsid surface depression described in several ENVs and rhi- noviruses (16) that has been reported to join different cel- Emerging Infectious Diseases • Vol. 9, No. 8, August 2003 939