Materials and Methods Clinical Specimens Study specimens consisted of 61 EV13 isolates and three cerebrospinal fluid (CSF) samples obtained as fol- lows. During 2000, the Enterovirus Laboratory (Service of Virology, National Microbiology Centre, Madrid) received 538 isolates for typing from different laboratories within Spain. These viruses were mostly recovered in a human rhabdomyosarcoma cell line with a positive cytopathic effect, which was confirmed by immunostaining with an anti-HEV group-specific antibody (Dako, Glostrup, Denmark). A neutralization test (Lim-Benyesh-Melnick immune serum pools) identified EV13 in 135 (25%) spec- imens. On the basis of their geographic and temporal dis- tribution (Figure 1), 61 isolates were selected for further study. The selected isolates were obtained from CSF (n=44), stool (n=12), and pharyngeal swab (n=5) samples from patients with aseptic meningitis, except for samples from seven patients who had fever, one who had acute flaccid paralysis, and two healthy persons (contacts of the patients with acute flaccid paralysis). The outbreak was carefully studied in the Canary Islands because of the high rate of infected patients and the context in which the cases occurred, including geographic location, climate, and tourism activity in the area during the year. Three addition- al CSF samples obtained from patients with aseptic menin- gitis in March 2000 were supplied by the Microbiology Diagnostic Service (National Microbiology Centre, Madrid, Spain). Amplification The VP1 3′ end genome region, successfully used to study the molecular epidemiology of other HEVs (7,12), was amplified in all 64 isolates. A 609-nucleotide (nt) frag- ment was amplified by using the RT-nested PCR method (10) with a total of 5 µ L of each 1:10 diluted cell culture isolate. A fragment of the expected length was also obtained in the three additional CSF samples, previously extracted with guanidinium thiocyanate lysis buffer (29). The VP1 3′ end fragment was useful in differentiating and studying the epidemiology of HEVs. However, since our main goal was to compare the sequences of the Spanish isolates with those of isolates from the rest of the world, we designed a specific PCR to amplify the VP1 5′ end gene, where a collection of isolates from Germany had been sequenced (22). For that purpose, specific primers of EV13 were designed by using the multiple alignments con- structed with the available EV13 sequences employing Clustal X software (free software available from: URL: http://www-igbmc.u-strasbg.fr/BioInfo/). The primer sequences were as follows: EV13_VP1sense (5′–3′) TGA- GACAGGGCACACATC; EV13_VP1anti (5′–3′) RESEARCH Figure 1. Temporal distribution of the Spanish echovirus 13 iso- lates 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. GCTAATGAATGGGATGGACAT. Its relative positions to EV11 polyprotein genome (X80059) were 2560–2577 and 2999–3019, respectively. Single-step RT and amplification was performed by using the Access RT-PCR kit (Promega Corp., Madison, WI). Five microliters of selected 1:10 diluted cell culture isolates was added to the reaction mix- ture which contained the following: 10 µ L of 5X reaction buffer; 2 mM magnesium sulfate; 250 µ M each of dATP, dCTP, dGTP, and dTTP; 0.4 µ M of each EV13_VP1sense and EV13_VP1 anti-primers; 1 U of avian myeloblastosis virus RT; and 1 U of thermus flavus DNA polymerase and Rnase-free distilled water to a final volume of 50 µ L. Amplification was performed in a PTC-200 Peltier thermal cycler (M.J. Research, Inc., Waltham, MA), programmed for a first RT step of 45 min at 48°C, followed by 2 min at 94°C, and for 45 cycles of 30 sec for denaturation at 94°C, 2 min for annealing at 60°C, and 30 sec for elongation at 68°C. Elongation was extended for 5 additional minutes in the last cycle. PCR products were detected by elec- trophoresis on 2% agarose gels stained with ethidium bro- mide. A 460-nt fragment was amplified in 14 representa- tive selected Spanish isolates. Sequencing and Sequence Analysis Cycle sequencing reactions of products from both RT- PCR assays were performed by using the Big Dye termi- nator kit (Applied Biosystems, Foster City, CA). Ambiguities were resolved by sequencing both sense and antisense strands. The 423-nt VP1 3′ end fragment (nt 2912–3334, according to the EV11 polyprotein genome, X80059) was analyzed through the multiple sequence comparison of the 64 viral sequences from Spain, the EV13 and ENV69 pro- Emerging Infectious Diseases • Vol. 9, No. 8, August 2003 935