Toscana virus (TOSV, Phlebovirus, family Bunyaviridae) infection is one of the most prevalent arboviruses in Spain. Within the objectives of a multidisciplinary network, a study on the epidemiology of TOSV was conducted in Granada, in southern Spain. The overall seroprevalence rate was 24.9%, significantly increasing with age. TOSV was detected in 3 of 103 sandfly pools by viral culture or reverse transcription–polymerase chain reaction from a region of the L gene. Nucleotide sequence homology was 99%-100% in TOSV from vectors and patients and 80%-81% compared to the Italian strain ISS Phi.3. Sequencing of the N gene of TOSV isolates from patients and vectors indicated 87%-88% and 100% homology at the nucleotide and amino acid levels, respectively, compared to the Italian strain. These findings demonstrate the circulation of at least 2 different lineages of TOSV in the Mediterranean basin the Italian lineaqe and the Spanish lineage.
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Within the last decade, the emergence and reemergence of arthropodbome virus (arbovirus) infections has been a health problem worldwide. West Nile virus (WNV) infection is a seasonal epidemic in North America (1). In southern Europe, WNV infections (2-4), tickborne encephalitis (5), sandfly fever Sicilian virus (SFSV), sandfly fever Naples virus (SFNV), and Toscana virus (TOSV) infections have been reported in Mediterranean countries (6,7). In Spain, a multidisciplinary network, EVITAR, has been recently created to study arthropod- and rodentborne viral diseases. One of the objectives of the network is to study TOSV infections in Spain.
TOSV (genus Phlebovirus, family Bunyaviridae) is an important agent of acute meningitis and meningoencephalitis in residents and visitors from Mediterranean countries (7-13). Aside from TOSV, other sandfly fever viruses, i.e., SFSV and SFNV, cause a brief, self-limiting febrile illness (6). Although TOSV is not normally associated with mild disease, serologic studies report high seroprevalence rates in areas of confirmed TOSV infections (7,14,15). Furthermore, a case of influenzalike illness caused by TOSV has recently been reported (16). In Spain, the first TOSV infections involving the central nervous system were reported in Granada in 1988 (7). Later, cases of TOSV infections were detected in other areas of Spain (15). Phylogenetic analysis of short polymerase chain reaction (PCR) products from the L segment showed that nucleotide sequences of TOSV isolates from Granada differ significantly from the Italian strain ISS Phl.3 (17).
TOSV was first isolated in Italy from the sandfly Phlebotomus perniciosus and later from P. perfiliewi (8,18). P. perniciosus is the most abundant anthropophilic species of Phlebotomus in Spain (19). The maximum activity of sandfly vectors for TOSV occurs during summer, along with most cases of TOSV infection (7). Vector-based TOSV surveillance is useful in reporting virus activity. It provides predictive indicators of transmission activity level associated with elevated human risk. However, no data are available on detection of TOSV from vectors in Spain.
As part of the study of TOSV infection within the aims of the EVITAR network, this work focused on 3 main objectives. First, a seroprevalence study to detect TOSV immunoglobulin G (IgG) antibodies was conducted. Second, by means of viral culture and reverse transcription (RT)-PCR, we investigated the presence of TOSV in pools ofphlebotomine sandflies. Finally, positive pools and viral isolates were phylogenetically characterized.
Materials and Methods
Prevalence Study of Anti-TOSV IgG Antibodies
Population Study for Selecting Participants
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 1845 years, and 15% were >65 years of age.
[FIGURE 1 OMITTED]
Serum Samples
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.
Investigation of TOSV in Vectors
Capture of Phlebotomine Sandflies
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 sand flies 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 RTPCR. Taxonomic classification of sandflies was carried out according to Gil-Collado et al. (19).
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