Molecular Evidence Indicates That Phlebotomus majorsensu lato (Diptera: Psychodidae) Is the Vector Species

of the Recently-Identified Sandfly Fever SicilianVirus Variant: Sandfly Fever Turkey Virus

Koray Ergunay,1 Ozge Erisoz Kasap,2 Zeliha Kocak Tufan,3 Mahur H. Turan,4 Aykut Ozkul,4 and Bulent Alten2

Abstract

Sandfly fever turkey virus (SFTV) is a recently-discovered sandfly fever Sicilian virus (SFSV) variant (familyBunyaviridae, genus Phlebovirus), characterized during retrospective evaluation of febrile disease outbreaks inTurkey. In addition to causing sandfly fever, SFTV was observed to induce elevation of liver enzymes, and tocause thrombocytopenia in affected individuals. This study was conducted to identify vectors for phlebovirusesincluding SFTV in Ankara province, Turkey, where evidence indicates ongoing circulation of SFTV, as well asToscana virus. Sandfly sampling was performed in Ankara province in the vicinity or in animal housing facilitiesin 15 peri-domestic sites. Male sandflies were identified morphologically, whereas females were evaluatedindividually for Phlebovirus RNA via a nested-PCR assay with consensus primers. Selected individuals and PCR-positive sandflies were subjected to barcoding via cytochrome c oxidase sequence analyses. The source of bloodmeals in virus-infected sandflies was investigated using a multiplexed PCR targeting the mitochondrial cyto-chrome b gene of various vertebrates. A total of 667 sandflies were captured in 11 locations. Morphologicalidentification of males (n = 226) revealed Phlebotomus major sensu lato as the most abundant species (38.9%),followed by Phlebotomus sergenti (20.4%), Phlebotomus halepensis (17.7%), Phlebotomus papatasi (10.2%), Phlebotomussimici (3.98%), Larrousius spp. (3.53%), Phlebotomus tobbi (1.32%), Phlebotomus perfiliewi perfiliewi (1.32%), andothers. Virus sequences were detected in 3 (3/441) sandflies, two of which were characterized as P. major s.l. viabarcoding. The detected sequences in sandflies were identified as SFTV, and were identical or similar to se-quences from patients from the same area and the prototype SFTV strain. Bovine and human blood meals weredemonstrated in SFTV-infected sandflies. P. major s.l. has been identified as the vector species for SFTV. Bovidaeneed to be evaluated as probable amplifying hosts for SFTV.

Key Words: Phlebovirus—Sandfly fever Sicilian virus—Turkey—Vector.

Introduction

Sandfly fever is a self-limited febrile condition, alsoknown as the 3-day, papatacci, or phlebotomus fever. It is

an influenza-like but highly incapacitating disease withsymptoms including fever, malaise, myalgia, and retro-orbitalpain (Dionisio et al. 2003). The viruses responsible for sandflyfever are classified in the family Bunyaviridae, genus Phlebo-virus, which includes 37 viruses grouped into 9 species, and 16

additional viruses registered as tentative species (Nichol et al.2005). Major phleboviruses associated with human disease inthe Mediterranean countries include sandfly fever Sicilianvirus (SFSV) and Toscana virus (TOSV). SFSV is mainly re-sponsible for sandfly fever, whereas TOSV is also associatedwith central nervous system infections (Dionisio et al. 2003;Charel et al. 2005). Phleboviruses are transmitted to humansby the bite of phlebotomine sandflies (Diptera: Psychodidae),and seroreactivity in indigenous populations follows the

1Department of Medical Microbiology, Faculty of Medicine, Virology Unit, Hacettepe University, Ankara, Turkey.2Department of Biology, Division of Ecology, Faculty of Sciences, Hacettepe University, Ankara, Turkey.3Department of Infectious Diseases and Clinical Microbiology, MOH Ankara Training and Research Hospital, Ankara, Turkey.4Department of Virology, Ankara University Faculty of Veterinary Medicine, Ankara, Turkey.

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distribution of vectors. The phlebotomus species identified asthe major vector for SFSV is Phlebotomus papatasi, whereasTOSV transmission is mainly via Phlebotomus perniciosus andPhlebotomus perfiliewi (Mertz 1997, Dionisio et al. 2003).

In addition to the SFSV and TOSV, new phlebovirus strainshave been identified in sandflies since 2006, including Mas-silia virus in southern France (Charel et al. 2009), Granadavirus in Spain (Collao et al. 2010), Punique virus in Tunisia(Zhioua et al. 2010), Adria virus in Albania (Papa et al. 2011),and novel sequences in Algeria (Moureau et al. 2010). Al-though serologic data indicate human and/or animal expo-sure to some of these strains, their pathogenicity has not yetbeen elucidated, and no association with clinical disease couldbe established. However, another new SFSV variant, sandflyfever Cyprus virus (SFCV), which has been identified in Greektroops in Cyprus, is clearly associated with outbreaks of fe-brile disease (Papa et al. 2006).

Sandfly fever turkey virus (SFTV) is a recently-discoveredphlebovirus variant that was characterized during retro-spective evaluation of outbreaks in 2007–2008 from Ankara(Central Anatolia), Izmir (Aegean coast), and Adana (Med-iterranean coast) provinces in Turkey (Carhan et al. 2010).SFTV is provisionally included in the sandfly fever Sicilianserocomplex, and displays 98% and 91.8% nucleotidehomology in the S segment to SFCV and SFSV prototypestrain Sabin, respectively (Carhan et al. 2010). In addition tothe common symptoms of sandfly fever, SFTV infectionswere frequently aggravated by gastrointestinal symptomsand elevation of liver enzymes, which has also been docu-mented for SFCV (Konstantinou et al. 2007), along withthrombocytopenia (Carhan et al. 2010; Kocak Tufan et al.2011). A seroprevalence study has demonstrated SFTV ex-posure in asymptomatic blood donors in Ankara and Konyaprovinces (Ergunay et al. 2011). Moreover, acute SFTV in-fections were also identified in Ankara in 2009, which indi-cates ongoing circulation of the virus in this region (KocakTufan et al. 2011).

The activity of various species of phlebotomine sandflieshas been observed in the Mediterranean, Aegean, and CentralAnatolian regions of Turkey, including P. papatasi andP. perfiliewi, well-known vector species for SFSV and TOSV,respectively (Yagci et al. 1998; Yaman and Dik 2006; Simseket al. 2007). The aim of this study was to perform a field studyto investigate sandflies as phlebovirus vectors in Ankara,where TOSV and SFTV cases have recently been reported(Ergunay et al. 2011; Kocak Tufan et al. 2011).

Materials and Methods

Setting and sample collection

The entomological survey was conducted in MamakCounty of Ankara province. Ankara (39�56’N, 32�52’E) is thecapital and second most densely-populated city in Turkey,with a population over 4.6 million. It was observed that themajority of SFTV-infected individuals from Ankara resided inthe same suburb, the Mamak county, hence the syndrome wasinformally named ‘‘Mamak fever’’ (Carhan et al. 2010; KocakTufan et al. 2011). Mamak (478 km2, elevation 899 m) is one ofthe largest rural counties of Ankara, with over 503,000 in-habitants in the northeastern part of the city.

For the sandfly sampling in or near Mamak County, 15locations were selected. Sampling was performed for three

consecutive days each week during July 2011. CDC miniaturelight traps ( John W. Hock Company, Gainesville, FL) equip-ped with an ultra-fine mesh were employed in each station.The traps were placed 1–2 meters above ground in the vicinityor in animal housing facilities (cow barns) in peri-domesticsites, and kept on site from late afternoon until dawn. Cap-tured sandflies were collected in the morning, placed indi-vidually into 1.5-mL microcentrifuge tubes according togender and trapping location, and transferred to the labora-tory on ice. The head and genitalia of male sandflies weredissected and visualized on slides prepared with Swan solu-tion, and were examined for morphological identification tospecies level via published keys (Theodor 1958; Artemiev1980; Lewis 1982). The remaining body parts were stored inabsolute ethanol until DNA extraction. Females were keptat - 80�C for further analysis.

Detection and characterization of phlebovirussequences in sandflies

Individual female sandflies were homogenized andclarified by centrifugation as described previously (Charelet al. 2009; Zhioua et al. 2010). Two hundred microlitersof the supernatant was used for RNA isolation using theHigh Pure Viral Nucleic Acid Kit (Roche Diagnostics,Mannheim, Germany). Ten microliters from the elutedRNA was used for reverse transcription via randomhexamers employing the RevertAid First Strand cDNASynthesis Kit (Fermentas, Vilnius, Lithuania). All com-mercial assays were performed according to the manu-facturers’ recommendations.

For the detection of phlebovirus sequences in individualsandfly homogenates, consensus primers targeting the viralpolymerase in the L segment of the viral genome (NPhlebo1 + /1 - and 2 + /2 - ) were used in a nested-PCR reaction, asdescribed previously (Sanchez-Seco et al. 2003), with 2.5 UHot Start Taq DNA polymerase (Bioron GmBH, Munich,Germany), in a PTC-200 Thermal Cycler (MJ Research,Waltham, MA). TOSV ISS.Phl.3 isolate, grown on Vero cells asdescribed previously (Ergunay et al. 2011), was used asa positive control, and extreme care was taken to preventcarry-over contamination. The amplicons were visualizedunder ultraviolet light after electrophoresis in 2% agarose gelsfor the expected amplicon size of 244 bases. Detected ampli-cons from the second round of PCR were cut out and purifiedwith the AXYprep DNA gel extraction kit (Axygen Bio-sciences, Union City, CA), ligated to the pJET1.2 vector sup-plied in the CloneJet PCR Cloning Kit (Fermentas), and wasused to transform cells as directed by the manufacturer. For-ward and reverse primers provided for sequencing wereemployed for the characterization of cloned amplicons usingan ABI Prism 310 Genetic Analyzer (Applied Biosystems,Foster City, CA). The obtained sequences were submitted toGenBank.

Barcoding and blood meal analysis in sandflies

For the characterization of the sandflies, the cytochrome coxidase I (COI) gene, frequently used for biological barcoding,was employed as described previously (Folmer et al. 1994). A658-basepair (bp) fragment of the COI gene was amplified viaLCO1490 and HCO2198 primers. PCR products were purifiedusing the QIAquick PCR product purification kit (Qiagen,

Phlebotomus major sensu lato AS SFTV VECTOR 691

Hilden, Germany), and subjected to cycle sequencing with theamplification primers from both directions.

Phlebovirus PCR-positive sandflies were further evaluatedvia a multiplexed PCR based on a mitochondrial cytochromeb gene to identify mammalian blood meals employing hu-man, dog, cow, and goat forward, with a universal reverseprimer, as previously described (Kent and Norris 2005). Theproducts, visualized in 2% agarose gels, were evaluated forblood meals from humans (334 bp), cows (561 bp), dogs(680 bp), and goats (132 bp), according to amplicon size.

Sequence data analysis

Sequences were edited using BioEdit Sequence AlignmentEditor and Chromas (version 2.13) software. The respectivesequences and similar sequences available in GenBank were

aligned with ClustalW. To construct taxon identity trees,neighbor joining (NJ) and maximum composite likelihoodanalyses were performed under the assumptions of Kimura’stwo-parameter model, and bootstrapping of 10,000 data ma-trices in MEGA 5.0 (Tamura et al. 2011).

Results

Species distribution of phlebotomines

A total of 667 sandflies were captured in 11 of 15 locations(Fig. 1); 226 (33.9%) were male, and 441 (66.1%) were female(Table 1). No trapping was observed in 4 locations despitefunctioning traps. Morphological identification of the malephlebotomines revealed P. major s. l. as the most abundantspecies (88/226, 38.9%), followed by P. sergenti (46/226, 20.4%),P. halepensis (40/226, 17.7%), P. papatasi (23/226, 10.2%), P.

FIG. 1. Map of Ankara province illustrating Mamak County and the sandfly sampling sites.

692 ERGUNAY ET AL.

simici (9/226, 3.98%), Larrousius spp. (8/226, 3.53%), P. tobbi (3/226, 1.32%), P. perfiliewi perfiliewi (3/226, 1.32%), Sergentomyiaspp. (2/226, 0.88%), Adlerius spp. (2/226, 0.88%), P. perfiliewigalilaeus (1/226, 0.44%), and Paraphlebotomus spp. (1/226,0.44%). The numbers and distribution of the sandfly speciesaccording to sampling location are provided in Table 1.

Identification and characterization of phlebovirussequences in phlebotomines

Three samples (3/441) were observed as reactive in thesecond round of nested PCR with phlebovirus consensusprimers NPhlebo 2 + and 2 - . The amplicons were ligated to

Table 1. The Number, Gender, and Species Distribution of Captured Sandflies

According to Sampling Location

Male Number Female Number Total

AI P. major s. l. 58 Phlebotomus spp.(39�54’09.79’’N, P. sergenti 532�54’19.58’’E) P. simici 2

P. tobbi 1Paraphlebotomus spp. 1Adlerius spp. 1Larroussius spp. 1Total 69 (46%) 81 (54%) 150

AII P. sergenti 35 Phlebotomus spp.(39�54’11.84’’N, Larroussius spp. 332�54’07.23’’E) P. major s. l. 2

P. halepensis 2Sergentomyia spp. 2P. papatasi 1P. simici 1P. perfiliewi galilaeus 1Total 47 (26.7%) 129 (73.3%) 176

AIII P. sergenti 3 Phlebotomus spp.(39�54’05.51’’N, P. major s. l. 232�54’18.73’’E) P. tobbi 2

P. papatasi 2P. simici 1Total 9 (37.5%) 15 (62.5%) 24

AIV P. papatasi 1 Phlebotomus spp.(39�54’03.52’’N, P.simici 132�54’17.59’’E) Total 2 (20%) 8 (20%) 10

AVI P. papatasi 3 Phlebotomus spp.(39�54’03.38’’N, P. sergenti 232�54’17.78’’E) Total 5 (31.3%) 11 (68.7%) 16

AVII P. papatasi 1 Phlebotomus spp.(39�53’08.53’’N,32�54’39.08’’E) Total 1 (0.7%) 145 (99.3%) 146

AVIII P. halepensis 7 Phlebotomus spp.(39�53’31.52’’N, P. major s. l. 632�54’35.33’’E) P. simici 4

P. perfiliewi perfiliewi 3Larroussius spp. 3P. papatasi 2P. sergenti 1Adlerius spp. 1Total 27 (90%) 3 (10%) 30

BI P. halepensis 20 Phlebotomus spp.(39�50’01.02’’N, P. papatasi 1432�56’46.16’’E) P. major s. l. 13

Total 47 (63.5%) 27 (36.5%) 74

BII P. halepensis 11 Phlebotomus spp.(39�50’03.85’’N, P. major s. l. 332�57’11.21’’E) Larroussius spp. 1

Total 15 (44.1%) 19 (55.9%) 34

BIII P. major s. l. 4 Phlebotomus spp.(39�49’47.60’’N,32�56’11.51’’E) Total 4 (57.2%) 3 (42.8%) 7

226 (33.9%) 441 (66.1%) 667

Phlebotomus major sensu lato AS SFTV VECTOR 693

cloning vector as described above, and 8–10 clones carryingthe insert were sequenced. Identical sequences of 204 bp (ex-cluding the primers) were retrieved from all clones analyzedfrom each sample. BLAST and BLASTX searches in GenBankrevealed these sequences to be 98–99% identical to SFTV.Multiple sequence alignment and phylogenetic analysis withsimilar and divergent phlebovirus sequences from GenBank

provided further support for the identification (Fig. 2). Iden-tical sequences were retrieved from sandflies captured at thesame location (GenBank accession numbers JN907011 andJN907013; Table 2), which were 1% divergent from the pro-totype SFTV sequence (GQ847513) in the maximum com-posite likelihood analysis. The sequence obtained at locationA1 ( JN907012) was found to be 1% and 2% divergent from the

FIG. 2. Phylogenetic analysis of the partial sequences of the polymerase gene segment detected in sandflies and patientsduring July–August 2011 in Ankara province, Turkey. GenBank accession numbers JN907007 to JN909012 represent thesequences included in this study, and GQ847513 is the prototype strain (SFTV, sandfly fever turkey virus; SFCV, sandflyfever Cyprus virus; SFSV, sandfly fever Sicilian virus; TOSV, Toscana virus; RVFV, Rift Valley fever virus).

694 ERGUNAY ET AL.

sequences from location AII and prototype SFTV, respectively.To provide evidence for the similarity between SFTV in vec-tors and human infections, sequences detected in sera of threeindividuals diagnosed with acute SFTV infections in August2011 in Ankara were included in the analyses ( JN907007,JN907008, and JN907010; Fig. 2). These sequences were ob-tained via the same methodology used for the phlebotomines,as described above. Maximum composite likelihood analysisrevealed identical sequences in one patient and sandflies fromlocation AI, and 98–99% similarity was observed for the re-maining sequences (data not shown). No relevant epidemio-logical connection could be demonstrated between patientsand the sampling sites other than being located in the sameneighborhood within an area of 5–7 km2.

Molecular characterization of Phlebotomus speciesdetected as phlebovirus vectors

A total of 26 sandflies were captured at the locations(AI and AII) and dates ( July 4–6) that included phlebovirus-positive samples (Table 2). Morphological examination of themales (n = 13) revealed eight specimens as P.major s.l., fourspecimens as P. sergenti, and one specimen as P. papatasi. Forone female specimen that was reactive in phlebovirus con-sensus PCR, no amplification could be seen despite repeatedtests (Table 2). The COI gene regions of the remaining females(n = 12) unidentified morphologically were subjected to se-quencing, along with three randomly-selected P. papatasi andP. major s.l. male specimens from different dates. The align-ment, including these specimens using the older GenBankentries, and those obtained for several Turkish sandfly species(Erisoz Kasap and Alten, unpublished data) as reference se-quences, was 454 bp long. Based on their COI sequences, eightfemales (including two infected ones) were assigned asP. major s.l., since seven of them were found to have identicalsequences with the sympatric P. major s.l. males, and onediffered by only five mutations. The NJ analysis also indicatedthat the rest of the females were clustered with the onlyP. sergenti sequence (FJ196442.1) available in GenBank, al-though they fell into two well-supported distinct lineages,which probably reflected intra-species variations (Fig. 3).

Blood-fed individuals were observed in 13.2% (58/441) ofthe female sandflies prior to homogenization, including allphlebovirus-positive specimens. Analysis via multiplexedPCR in these samples revealed bovine blood meals in all andhuman blood meals in two samples captured in the samelocation (Table 2).

Discussion

Despite serologic evidence for exposure, detection of acuteinfections, and documentation of the presence of various

species of phlebotomine sandflies, vector surveillance forphleboviruses in Turkey has not been performed previously(Ergunay et al. 2010). Since SFTV is associated with a moresevere clinical picture than the typical sandfly fever symp-tomatology, the identification of phlebotomine species asvectors and their associated habitats are of substantial im-portance. This study was conducted to identify phlebovirusesin sandflies in Ankara province, where SFTV outbreaks wereobserved from 2007 to 2010, indicating continuous circulationof the virus, in addition to TOSV cases detected in 2010, aswell as documented exposure in blood donors (Ergunay et al.2011, 2011; Kocak Tufan et al. 2011).

Throughout July 2011, we captured 667 sandflies in 11sampling locations (Table 1). Morphological identification ofthe male phlebotomines indicated that the most frequently-observed species were P. major s.l. (38.9%), P. sergenti (20.4%),P. halepensis (17.7%), and P. papatasi (10.2%), followed byothers. The abundance of P. major s.l. in the sampling area isfurther supported by the COI barcoding of females, whichindicated that this species constituted 61.5% of the sandfliesinvestigated, despite a limited number of evaluated speci-mens. Only one previous report on the species distribution ofphlebotomines in Ankara province and the surrounding areaexists (Yagci et al. 1998). In that study, the predominant spe-cies was P. perfiliewi, as identified in 98.4% of the 2248 sand-flies collected from 1994–1995. However, sandfly samplingwas performed in different locations (Cayirhan, Cubuk, andKazan counties, 35–100 km north-northeast of the currentsampling sites), and only one location was a peri-domesticsite, where P. perfiliewi was accompanied by P. simici, P. ser-genti, P. papatasi, and other species. Nevertheless, P. perfiliewiwas the only phlebotomine species detected in the rest of thesampling locations, which were agricultural sites with nearbyanimal shelters, distant from human residential areas (Yagciet al. 1998). The frequency of P. perfiliewi and its subspecies inour study was low (4/226, 1.76%). The female predominanceobserved in our survey (66.1% females) was also noted in thereport by Yagci’s group (89.1% females). Since our field sur-vey was confined to the neighborhoods associated with SFTVemergence, it remains to be determined whether the sandfliescaptured in this study represent the currently dominantsandfly species present in Ankara province.

The captured female sandflies were processed individuallyfor the detection of phlebovirus RNA via a nested-PCR assaywith degenerated primer pairs targeting the polymerase (L)segment of the viral genome (Sanchez-Seco et al. 2003). Theseprimers are frequently used in field studies, and enables thedetection of new strains, which was the case for SFTV (Carhanet al. 2010). Three sandfly specimens captured inside two cowbarns (locations AI and AII) were found to possess viral RNAidentified as SFTV via sequencing (Table 2). Moreover, the

Table 2. Features of Phlebovirus Consensus PCR-Reactive Samples

No. Sampling location Date collected Sequence ID Host Blood meal

1. AI(39�54’09.79’’N, 32�54’19.58’’E) July 4–6 SFTV ( JN907012) Phlebotomus sp. Yes (Bovine)2. AII(39�54’11.84’’N, 32�54’07.23’’E) July 4–6 SFTV ( JN907011) P.major s.l. Yes (bovine and human)3. AII(39�54’11.84’’N, 32�54’07.23’’E) July 4–6 SFTV ( JN907013) P.major s.l. Yes (bovine and human)

Phlebotomus major sensu lato AS SFTV VECTOR 695

FIG. 3. Neighbor joining tree based on the partial cytochrome c oxidase I (COI) sequences used for barcoding of sandflies.AI and AII indicate the sampling locations (see text for details). Phlebovirus consensus PCR-positive samples are indicatedwith an asterisk.

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sequences detected in sandflies during the field survey wereidentical to or very similar to SFTV sequences obtained frompatient samples (Fig. 2), which further supports circulation ofthe SFTV in the region, and symptomatic human infectionscaused by this strain. Interestingly, PCR-positive SFTV pa-tients were seen in early to mid-August (data not shown),while SFTV in vectors could be identified only during earlyJuly, and samples from the same locations were negativethrough middle to late July. The implications of this obser-vation, whether coincidental or not, are not clear. For a com-prehensive analysis of virus circulation dynamics in theregion, SFTV surveillance needs to performed throughout thesandfly season to reveal periods of peak activity, which wasnot possible in our study.

The phlebovirus infection rate in phlebotomines observedin this study (3/441, 0.68%) is comparable with previous re-ports (Moureau et al. 2010; Zhioua et al. 2010). Two of thethree SFTV-infected sandflies were characterized as P. majors.l. via COI barcoding, while another phlebotomus withpositive PCR could not be characterized via this method,presumably due to the presence of inhibitors, and could onlybe identified to the genus level (Table 2). Nevertheless, com-pletely-characterized samples and the overall species distri-bution of sandflies in the field strongly suggest P.major s.l. asthe vector species responsible for SFTV transmission. AnotherSFSV variant, Corfou virus, which is distantly related to SFTV(Fig. 2), has been detected in P. major s.l. sandflies as well(Rodhain et al. 1985). In addition to P. papatasi and P. major s.l.,phleboviruses belonging to the Sicilian serocomplex have alsobeen identified in P. ariasi (Izri et al. 2008), P. perniciosus,P. longicuspis, and Sergentomyia minuta (Zhioua et al. 2010),demonstrating the adaptation capability and wide distribu-tion of these agents.

The evaluation of SFTV-infected sandflies individually viamultiplexed PCR targeting the mitochondrial cytochrome bgene from various vertebrates enabled direct identification ofthe sources of blood meals. All virus-positive samples werefound to be reactive for blood of bovine origin, which is notsurprising since all were captured inside cow barns withlivestock nearby. Two samples originating from location AIIhad blood meals from humans as well (Table 2), establishinganother link between the virus, the vector, and human ex-posure in the region. The potential animal reservoirs forphleboviruses have not been studied in detail. Althoughpreliminary findings suggested some species as candidates,the role of animals in virus survival and propagation isthought to be of lesser importance, due to the effectivetransovarial and venereal transmission of the virus in vectors(Tesh and Modi 1987; Tesh et al. 1992). In a recent report onTOSV exposure in animals, seroprevalence rates of 48.3% indogs, 17.7% in goats, 32.3% in sheep, 59.6% in cats, 17.9%in cows, 22% in pigs, and 64.3% in horses were described(Navarro-Mari et al. 2011). However, virus replicationcould only be detected in goat serum via PCR, suggesting thatthese animals are amplifying hosts for the virus rather thanreservoirs. Further studies are required to explore cows andother animals as possible amplifying hosts for SFTV or otherviruses.

In conclusion, P. major s.l. has been identified as the vectorspecies for the recently-discovered SFSV variant SFTV, whichis associated with febrile disease with elevated liver enzymesand gastrointestinal symptoms in humans.

Acknowledgments

The authors are grateful to Irfan Atmaca and Salim Calis fortechnical assistance, and N. Emin Guven for graphics.

Author Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Koray Ergunay

Hacettepe University Faculty of MedicineDepartment of Microbiology and Clinical Microbiology

Virology UnitMorphology Building, 3rd floor

06100 Sihhiye, AnkaraTurkey

E-mail: ekoray@hacettepe.edu.tr

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