RESEARCH Molecular Analysis of Echovirus 13 Isolates and Aseptic Meningitis, Spain Ana Avellón,* Inmaculada Casas,* Gloria Trallero,* Carmen Pérez,† Antonio Tenorio,* and Gustavo Palacios‡ Echovirus 13 (EV13), considered rare, was reported worldwide in 2000, mostly related to aseptic meningitis out- breaks. In Spain, 135 EV13 isolates were identified. The genetic relationships between 64 representative strains from Spain and other reported isolates from the United States, Germany, Italy, Japan, and Sweden were described by analyzing the partial sequence of the major capsid pro- tein (VP1) gene. The strains from Spain were clearly iden- tified as EV13 (79.5% similarity with the EV13 reference strain) and were grouped phylogenetically into two different clusters (by origination on either the Iberian Peninsula or Canary Islands). Isolates from Germany from 2000 clus- tered with the Canary Islands group. The isolates from other countries obtained before 2000 were genetically dis- tant. Changes in EV13 coding sequence involved several differences in the C-terminal extreme of the VP1 protein. Part of the neutralizing antigenic site III has been described in this genome region in poliovirus and swine vesicular dis- ease virus. Tgenus, Picornaviridae family) are common human he human enteroviruses (HEVs; Enterovirus [ENV] pathogens with a worldwide distribution. HEVs are found in temperate climates and especially in the tropics because warm weather favors their spread. Humans are the only known reservoir for HEV, and the main transmission route is fecal-oral. HEV infections may be unapparent or related to different disorders, including encephalitis, pleurodynia, myocarditis, conjunctivitis, or systemic infections in neonates. These viruses are commonly reported in associ- ation with aseptic meningitis outbreaks in pediatric patients (1). Although five HEV species exist (2), they are grouped into four clusters (HEV-A, -B, -C, and -D) on the basis of sequence analysis of the major capsid protein (VP1) and the 3′ noncoding region (3). The HEV-B cluster includes coxsackie virus B (CBV), coxsackie virus A9, ENV69, and all echoviruses (EV). The advent of nucleic acid amplification methods has *Instituto de Salud Carlos III, Madrid, Spain; †Doctor Negrín Hospital, Las Palmas de Gran Canaria, Canary Island; and ‡Columbia University, New York, New York, USA facilitated the study of the molecular epidemiology of HEV (4–6). Several reverse transcription-polymerase chain reaction (RT-PCR) methods were developed for this purpose, most of which analyze different sequence frag- ments within the VP1 gene (7–9). Moreover, the amplifi- cation and subsequent analysis of the VP1 3′ end partial gene have been successfully used to serotype HEVs (10,11) and to describe the molecular epidemiology of EV30 (12). Analysis of this fragment of the enteroviral genome has permitted the study of important pathogenic structures, such as part of the antigenic sites described for polioviruses (13), CBV, and swine vesicular disease virus (SVDV) (14,15), and part of the canyon structure, related to viral attachment, which has been described in several HEVs and rhinoviruses (16). The EV strains most frequently isolated in patients with aseptic meningitis are EV30, EV6, EV11, and EV9 (1). Displacement of prevailing lineages on the basis of immune escape (antigenic drift) has been suggested for EV30 as the mechanism to maintain viral circulation lev- els that increase periodically (12,17,18). Unlike EV30, EV13 has been considered a rare virus (1). Until 1999, iso- lation data showed very low rates in countries such as the United States (19), Ireland (20), and England and Wales (21). During 2000, EV13 circulation, mostly related to aseptic meningitis, was reported in America (19), Europe (20,22–25), Asia (26), and Australia (27). In Spain, an out- break of aseptic meningitis attributable to EV13 occurred from February to October 2000 (25), the first identification of this virus in Spain since record keeping began in 1988 (28). We describe the molecular epidemiology of the EV13 isolates obtained in Spain from the 2000 outbreak by ana- lyzing the VP1 partial sequences of 64 identified viruses. We also compare them with other isolates for which sequences have been deposited in databases. Finally, by analyzing the molecular nature of their changes in nucleotides and amino acids, we attempt to describe the nature of, and reasons for, the actions and epidemiology of EV13. 934 Emerging Infectious Diseases • Vol. 9, No. 8, August 2003