The seroprevalence study was conducted on study participants from the Granada population. Participants were retrospectively selected on the basis of demographic data and estimations of seroprevalence rates to TOSV (7,15). To evaluate differences in seroprevalence rates within Granada, the province was divided into 5 geographic areas: urban, metropolitan, south, west/southwest, and north/northeast (Figure 1). By age groups, 20% were <18 years, 65% were 18–65 years, and 15% were >65 years of age.

Serum samples were collected from September to December 2003. Specimens from adults 18- to 65-years of age were collected from anonymous healthy blood donors. Specimens from persons <18 and >65 years of age were obtained from 2 laboratories in Granada from persons with noninfectious pathologic features. Only data on age, sex, and geographic area of origin were recorded from the study population. Anti-TOSV IgG was detected by a commercial enzymatic immunoassay, EIA Enzywell Toscana virus IgG (Diesse, Italy), following the manufacturer's instructions.

Phlebotomine sandflies were captured with CDC light traps (20) from June to October of 2003 and 2004. Traps were placed in 16 areas of the rural environment where the first cases of meningitis by TOSV appeared (21) (Figure 1). Sandflies were trapped after dusk until dawn. Traps were immediately transported to the laboratory to pool the individual vectors by sex and trapping area. In the 2003 season, pools of male sandflies were used for taxonomic classification, and pools of female sandflies were tested for TOSV by RT-PCR. In the 2004 season, up to 10% of individual sandflies were separated for taxonomic classification. The remaining insects were pooled by sex and trapping area and tested for TOSV by viral culture and RT-PCR. Taxonomic classification of sandflies was carried out according to Gil-Collado et al. (19).

Phlebotomines were introduced in vials with sterile crystal beads and 0.5 mL minimal essential medium supplemented with 20% fetal bovine serum and antimicrobial mix (0.4 mg/mL gentamicin, 0.5 mg/mL vancomycin, and 2.5 μg/mL amphotericin B). Vials were vortexed and centrifuged at 13,000 rpm for 5 min. A 200-μL aliquot of the supernatant was injected into tubes with African green monkey kidney cells; the remaining supernatant was frozen at –80°C. The pellet with the phlebotomines was used for RT-PCR. Tube cultures were incubated at 37°C and examined daily for the appearance of cytopathic effect (CPE). Tubes with positive CPE were tested for TOSV by RT-PCR.

Viral RNA from cell cultures was extracted by using QIAamp viral RNA kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. Viral RNA from sandflies was isolated by using the same kit, with minor modifications. Briefly, 500 μL lysis buffer was added to the tubes containing the phlebotomine sandflies, tubes were vortexed and centrifuged at 13,000 rpm for 10 min, and 250 μL of the supernatant was used for RNA extraction. A generic RT-nested-PCR method was used to detect TOSV RNA as described previously (17), which amplifies a 244-bp fragment of the L gene of phleboviruses. Sequences of the primers are shown in Table 1.

Specific TOSV detection was achieved by sequencing PCR products, as described previously (17). Briefly, PCR products were purified from a 2% low-melting-point agarose gel with the QIAquick PCR purification kit (Qiagen). Sequencing reactions were performed by using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA) and analyzed by an ABI model 377 automated sequencer (Applied Biosystems). Two sequencing reactions were carried out on each PCR product by using the sense and antisense primers of the nested-PCR step. TOSV-specific sequences were confirmed by BLAST (basic local alignment search tool) search against GenBank databases. Sequences of the L gene from positive pools were compared with the corresponding sequence of the Italian strain ISS Phl.3 and with 1 Spanish TOSV isolate (STI) obtained previously (17) with the ClustalW Multiple Sequence Alignment program (1.82 version; European Bioinformatics Institute, Cambridge, UK).

Phylogenetic analysis of the sequences from the partial L gene and the complete N gene was carried out with MEGA 3 program (22) by using the Kimura 2 parameter model for nucleotides and amino model with Poisson correction for amino acids to calculate distances between sequences with confidence values of 1,000 bootstrapping trials. Phylogenetic trees of the L gene were constructed from TOSV obtained from sandflies and previous STIs and from different phleboviruses. Available GenBank accession numbers of the phleboviruses sequences were TOSV ISS Phl.3: X68414; RVFV: X56464; UUKV: D10759; and phlebovirus Chios-A (Chios): AY293623. Phylogenetic trees of the N gene were constructed from STI sequences obtained from sandflies and patients and from the phleboviruses sequences described above.

TOSV sequences of the L (partial) gene from vectors and that from a human STI were almost identical. At the nucleotide level, 19%–20% diversity was observed between sequences obtained from Spanish samples and those of the Italian strain, whereas the homology at the amino acid level was almost 100% (Table 4). Phylogenetic analysis yielded the same results (Figure 2). The phylogenetic tree of nucleotide sequences show 1 group containing all TOSV, in which the Spanish ones form, with a bootstrap value of 100, a different cluster. This diversity is not reflected in the amino acid sequences since both the Spanish and Italian TOSV group into a unique cluster.

Nucleotide and amino acid sequence homology within the N gene was ≈98%–100% among STIs from vectors and patients. Compared with the Italian strain, a 100% homology at the amino acid level was observed between STIs and the Italian strain. However, nucleotide sequences showed a 12%–13% difference between the strains (Table 4). The phylogenetic tree of nucleotide sequences shows that, within the TOSV group, STIs form a different cluster. However, at the amino acid level, both STIs and Italian TOSV strain group into a unique cluster (Figure 3).