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Phlebotomine sand flies are known to transmit Leishmania, bacteria, and viruses that affect humans and animals in many countries worldwide. Sand fly-borne viruses belong mainly to the Phlebovirus, Vesiculovirus, and Orbivirus genera, and some of them are associated with outbreaks or sporadic human cases in the Mediterranean Europe. Up to now, Toscana virus is the only phlebovirus of medical importance identified in France. To study the diversity of the sand fly population living in the southeastern France, an entomological study was conducted from May to October, 2007. Most of the trapped sand flies belonged to Phlebotomus perniciosus (82.0%) and Sergentomyia minuta (17.3%) species; only three specimens were Phlebotomus ariasi. Molecular characterization of the P. perniciosus specimen based on the mitochondrial cytochrome b gene demonstrated different subpopulations living in the same areas. Most of the specimens belonged to the haplotypes pern01 and pern09, already described in France, but some belonged to original new haplotypes. The detection of one viral sequence clustering with Massilia/Granada virus, and of four sequences corresponding to two potential new phleboviruses (proposed names Olbia and Provencia viruses, respectively), revealed an unexpected diversity of phlebovirus species in France.

Sandfly fever turkey virus (SFTV) is a recently-discovered sandfly fever Sicilian virus (SFSV) variant (family Bunyaviridae, genus Phlebovirus), characterized during retrospective evaluation of febrile disease outbreaks in Turkey. In addition to causing sandfly fever, SFTV was observed to induce elevation of liver enzymes, and to cause thrombocytopenia in affected individuals. This study was conducted to identify vectors for phleboviruses including SFTV in Ankara province, Turkey, where evidence indicates ongoing circulation of SFTV, as well as Toscana virus. Sandfly sampling was performed in Ankara province in the vicinity or in animal housing facilities in 15 peri-domestic sites. Male sandflies were identified morphologically, whereas females were evaluated individually 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 blood meals in virus-infected sandflies was investigated using a multiplexed PCR targeting the mitochondrial cytochrome b gene of various vertebrates. A total of 667 sandflies were captured in 11 locations. Morphological identification 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%), Phlebotomus simici (3.98%), Larrousius spp. (3.53%), Phlebotomus tobbi (1.32%), Phlebotomus perfiliewi perfiliewi (1.32%), and others. Virus sequences were detected in 3 (3/441) sandflies, two of which were characterized as P. major s.l. via barcoding. The detected sequences in sandflies were identified as SFTV, and were identical or similar to sequences from patients from the same area and the prototype SFTV strain. Bovine and human blood meals were demonstrated in SFTV-infected sandflies. P. major s.l. has been identified as the vector species for SFTV. Bovidae need to be evaluated as probable amplifying hosts for SFTV.

Sandfly-transmitted phleboviruses, such as Toscana, sandfly fever Sicilian, and sandfly fever Naples, can cause human disease and circulate at high rates in Mediterranean countries. Previous studies have also established that viruses other than phleboviruses may be detected in and isolated from sand flies. The recent detection and isolation (in a large variety of mosquito species) of insect-only flaviviruses related to cell fusing agent virus has indicated that the latter is not an evolutionary remnant but the first discovered member of a group of viruses, larger than initially assumed, that has high genetic heterogeneity. Insect-only flaviviruses have been detected in and/or isolated from various species of mosquitoes, but nevertheless only from mosquitoes to date; other dipterans have not been screened for the presence of insect-only flaviviruses. The possible presence of flaviviruses, including insect-only flaviviruses, was investigated in sand flies collected around the Mediterranean during a trapping campaign already underway. Accordingly, a total of 1508 sand flies trapped in France and Algeria, between August 2006 and July 2007, were tested for the presence of flaviviruses using a PCR assay previously demonstrated experimentally to amplify all recognized members of the genus Flavivirus, including insect-only flaviviruses. Two of 67 pools consisting of male Phlebotomus perniciosus trapped in Algeria were positive. The two resulting sequences formed a monophyletic group and appeared more closely related to insect-only flaviviruses associated with Culex mosquitoes than with Aedes mosquitoes, and more closely related to insect-only flaviviruses than to arthropod-borne or to no-known-vector vertebrate flaviviruses. This is the first description of insect-only flaviviruses in dipterans distinct from those belonging to the family Culicidae (including Aedes, Culex, Mansonia, Culiseta, and Anopheles mosquito genera), namely sand flies within the family Psychodidae. Accordingly, we propose their designation as phlebotomine-associated flaviviruses.