Molecular analysis of echovirus 13 isolates and aseptic meningitis, Spain

Abstract

Echovirus 13 (EV13), considered rare, was reported worldwide in 2000, mostly related to aseptic meningitis outbreaks. 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 protein (VP1) gene. The strains from Spain were clearly identified 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 clustered with the Canary Islands group. The isolates from other countries obtained before 2000 were genetically distant. 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 disease virus.

Figure 1

Temporal distribution of the Spanish echovirus 13 isolates during 2000. The isolates were grouped attending to their geographic origin as from the Iberian Peninsula and from the Canary Islands. The number of viruses included in the study (in parentheses) as well as the total isolates per month are shown. *Includes three sequences obtained directly from cerebrospinal fluid.

Figure 2

Phylogenetic tree of the VP1 3′ terminal region, which identifies the Spanish isolates as echovirus 13 (EV13) and differentiates 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.

Figure 3

Histogram of the observed distances (Kimura two parameters 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 (light gray): mean 0.008 (standard deviation [SD] 0.004; n=351); Iberian Peninsula (dark gray): mean 0.010 (SD0.006; n=666). Distances between heterologous groups (black): mean 0.033 (SD 0.005; n=999).

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 isolates as well as the German (n=12), Italian (n=1), Swedish (n=1), and Japanese (n=2) ones. Phylogenetic tree reconstructed with the maximum-likelihood method, 50 Jumble. Gray sequences correspond to the 2001 isolates.

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 found in a similar location to the canyon structure of poliovirus as reported by (40).