sandflies were trapped during 4 days in july 2005 in 2 cities in southern france ( marseille and nice ) by using techniques previously reported ( 7 ) ( figure 1 ) . adult flies shelter during the day in dark , quiet , and humid places , and eggs are laid in terrestrial microhabitats rich in organic matter that provides food for larvae , including soils containing herbivorous animal feces . therefore , traps were placed at dawn in or near animal housing facilities ( horse stalls , rabbit hutches , hen houses ) or in areas where visceral canine leishmaniasis is endemic ( 8) . each morning , sandflies were collected and identified morphologically , by dissecting genital organs , according to morphologic taxonomic keys ( 9 ) . sandflies were pooled ( up to 30 individual flies ) according to trapping origin , species , and gender ( table ) in 1.5-ml tubes and processed as described ( 7 ) . cdc light traps , adapted for sandfly trapping , placed in horse stables ( a ) , near rabbit hutches and henhouses ( b ) , and in quiet places in the shade of human habitations , where dogs sleep ( c ) . engorged female phlebotomus perniciosus sandfly trapped in a horse stable ( d ) ; spermatheca of ph . a variety of primers targeting different genes were used : primers specific for tosv in the polymerase gene ( 10 ) and primers designed in this study from the alignment of nucleoprotein sequences of selected phleboviruses ( retrieved from genbank ) specific for the species sandfly fever naples virus ( sfnv - s1 [ 5-cttyttrtcytcyctrgtgaagaa-3 ] , sfnv - r1 [ 5-atgatgaagaaratgtcagagaa-3 ] , sfnv - s2 [ 5-gcrgccatrttkggyttttcaaa-3 ] , sfnv - r2 [ 5-cctggcagrgacacyatcac-3 ] ) . the reverse - transcription  pcr ( rt - pcr ) was performed by using the access rt - pcr kit ( promega , madison , wi , usa ) according to the manufacturer s recommendations . l and s rna primers were used at 0.8 mol and 0.4 mol per reaction , respectively . the cycling program of the rt - pcr reaction consisted of 48c for 45 min and 94c for 2 min , followed by 40 cycles at 94c for 30 s , the annealing temperature for 1 min , and 68c for 45 s , with a final elongation step at 68c for 7 min . using ground material obtained from pools ak and ar and patients samples , we injected vero - e6 cells ( protocol available on request ) . perniciosus species ( table , figure 1 ) , 483 ( 96.8% ) in marseille , 110 ( 62.5% ) in nice . mascitii ( n = 4 , 0.8% ) , and ph . ariasi ( n = 3 , 0.6% ) in marseille . in addition , 123 sergentomyia minuta were identified and led to the detection of tosv rna , as recently reported ( 7 ) . finally , 61 pools of phlebotomus spp . we detected tosv rna by both pcrs in 2 pools ( ak and ar ) of female ph . ak and ar sequences were distinct from each other ( 1.4% and 1% nucleotide divergence for s and l rna sequences , respectively ) , from other sequences available in genbank , and from those determined during this study or in previous studies ( 0.7%21.5% and 17.4% nucleotide divergence for s and l rna sequences , respectively ) ( 7 ) . sequences from 3 tosv - infected patients who lived in the region of marseille were included in the genetic analysis . for 2 patients , sequences were obtained after virus isolation in vero cells : strain imtssa/2004 ( genbank accession no . ay766034 ) ( 6 ) and strain 1500590 ( genbank accession no dq975232975233 , this study ) . another sequence was determined from direct amplification of a cerebrospinal fluid sample ( genbank accession no . nucleotide distances ( appendix figure ) clearly allowed discrimination of the 2 genotypes previously described as the italian and the spanish genotypes by reference to the origin of viruses . sequences obtained from sandfly and clinical material collected in france indicated that both genotypes are circulating in southern france . indeed , ph . perniciosus specimens ak and ar and human samples 5501805 and imtssa/2004 displayed high identity levels with tosv of spanish origin . in contrast , s. minuta specimen smin2005 and human sample 1500590 were more closely related to the italian strain iss.phl3 ( appendix figure ) . phylogenetic analysis of nucleotide sequences ( figure 2a ) showed that spanish , french , and italian tosv and sfnv formed a cluster that included members of the species sandfly fever naples virus , for which genetic data were available . perniciosus ( ak and ar ) and the 2 tosv sequences from clinical samples ( imtssa2004 and 5501805 ) grouped with spanish tosv ( 100% bootstrap support ) , while s. minuta specimen smin2005 and human sample 1500590 clustered with italian tosv sequences . topology analysis indicated that the 4 french tosv most closely related to spanish tosv root the spanish sequences ( figure 2a ) . further studies , with more sequences , are needed to investigate this finding . as previously reported ( 3 ) , despite significant variability between tosv isolates at the nucleotide level ( up to 13.2% ) , phylogenetic analysis of amino acid sequences did not allow discrimination between the italian and spanish genotypes ( appendix figure , figure 2b ) . indeed , in the nucleoprotein region considered for analysis , all tosv amino acid sequences were 100% identical , except that obtained from s. minuta , which diverged by 1 amino acid ( data not shown ) . phylogenetic trees based on nucleotide ( a ) and amino acid ( b ) sequences in the nucleoprotein gene of phleboviruses within the species sandfly fever naples virus . sequence information corresponds to virus / country of origin / strain / genbank accession no / host . sequence alignment was achieved with clustalx 1.81 with sequences from other phleboviruses retrieved from genbank . genetic distances were calculated with the pairwise distance and jukes - cantor methods at the nucleotide and amino acid level , respectively . this study provided genetic evidence , from the analysis of 2 independent genes , that tosv circulates in populations of ph . the absence of detection of tosv in the nice region may be due to the small number of sandflies collected . a similar study aimed at collecting a larger number of sandflies is therefore necessary to address whether tosv circulates in the region of nice and whether , if it does , it belongs to the spanish genotype , the italian genotype , or another yet unrecognized genotype . the rate of infection observed with tosv in sandflies collected during this study ( 2/675 ) is of the same magnitude of that reported in spain ( 3 ) and lower than rates reported in italy ( 2 ) . the distribution and respective frequency of sandfly species in france ( this study ) and spain ( 3 ) were different : ph . sergenti ( 8.5% ) were cataloged in spain , not in france , whereas ph . perfiliewi , known to replicate tosv in nature in italy , has not been identified in either french or spanish studies . in conclusion , we have provided the first evidence for the circulation of tosv in ph . we also determined that tosv belonging to the 2 genotypes previously recognized in italy and spain circulates and causes human infections in southeastern france . genetic distances in the nucleoprotein gene between viruses within the sandfly fever naples virus species . diversity was calculated by the pairwise - distance algorithm implemented in the mega software program . ( reference : kumar s , tamura k , nei m. mega3 : integrated software for molecular evolutionary genetics analysis and sequence alignment .
toscana virus ( tosv ) , an arthropodborne phlebovirus transmitted by sandflies , can cause febrile illness and meningitis . 
 the vector of tosv in france was unknown . 
 we detected tosv rna in 2 ( female phlebotomus perniciosus ) of 61 pools of sandflies captured in southeastern france . 
 two genotypes of tosv were identified .