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Background: Phlebotomine sandflies are vectors of several pathogens with significant impact for public health. This study was conducted to investigate and characterize phlebovirus and Leishmania infections in vector sandflies collected in the eastern Thrace region in Turkey and Northern Cyprus, where previous data indicate activity of these agents., Methods: Field sampling of sandflies was performed at 4 locations in Edirne and Tekirdag provinces of eastern Thrace and at 17 locations in Lefkosa, Girne, Magosa and Guzelyurt provinces of northern Cyprus. In sandfly pools, phlebovirus RNA and Leishmania DNA were screened via a generic polymerase chain reaction (PCR) and kinetoplast minicircle PCR, respectively. Selected sandfly specimens unsuitable for pathogen detection were identified to species level. Cytochrome oxidase 1 gene region was used for DNA barcoding of selected specimens and pathogen positive pools. Positive amplicons were cloned and characterized by sequencing., Results: A total of 2690 sandflies, collected from Eastern Thrace (15.4%) and Northern Cyprus (84.6%) were evaluated. Morphological examination of 780 specimens from Cyprus exhibited Phlebotomus perfiliewi sensu lato (72.6%), Phlebotomus tobbi (19.7%), Phlebotomus papatasi (2.8%), Laroussius sp. (1.6%) and Sergentomyia azizi (1.6%), Sergentomyia sp. (0.9%), Sergentomyia minuta (0.5%) and Phleobotomus jacusieli (0.1%) species. Pathogen screening was performed in 1910 specimens distributed in 195 pools. In eight pools of P.tobbi sandflies collected in Cyprus, Leishmania infantum DNA was demonstrated. Toscana virus (TOSV) genotype A sequences were identified in two pools of P. perfiliewi s.l. and one pool of P.tobbi sandflies from Cyprus. Co-infection of TOSV and Leishmania infantum was characterized in a P.tobbi pool. Sequences belonging to novel phleboviruses are revealed in three P. perfiliewi s.l. pools. One sequence, provisionally named Edirne virus, identified in Edirne province in eastern Thrace, demonstrated the highest rate of genomic similarity to Adria and Salehabad viruses. Furthermore, Girne 1 and Girne 2 viruses, identified in Girne province, revealed similarities to TOSV and Sandfly Fever Sicilian virus and related strains, respectively., Conclusions: Activity of TOSV genotype A strains in Cyprus and co-infection of sandfly vectors with L. infantum was documented for the first time. Novel phlebovirus strains of unknown medical significance was identified in sampling regions.

Introduction: Arbovirus screening in invertebrate vectors is an important component of the vector-borne disease surveillance programs. Turkey has been shown to harbor medically important mosquito-borne arboviruses such as West Nile Virus (WNV). However, limited information about infections in vectors are currently available. This study was performed to provide preliminary data from Eastern Thrace region, Turkey, where no arbovirus vector surveillance has previously been performed., Materials and Methods: Mosquito sampling was undertaken at 23 sites in Edirne province during July, 2012. All specimens were identified morphologically, and selected individuals were subjected to DNA barcoding via cytochrome c oxidase I (COI) sequencing. Consensus PCR for Flavivirus, Alphavirus, and Phlebovirus genera and WNV-specific nested and real-time reverse transcription PCR were employed for mosquito pool screening and/or confirmation. Viral sequences detected in pools were characterized via sequencing., Results: A total of 9261 mosquitoes were captured and distributed into 232 pools from the following species: Ochlerotatus caspius (90.9%), Culex pipiens sensu lato (s.l.) (4.7%), Anopheles pseudopictus (3%), and Anopheles maculipennis s.l. (1.3%). Specimens morphologically classified as Cx. pipiens s.l. were identified as Cx. pipiens pipiens via barcoding. Thirty-seven mosquito pools (15.9%) were positive in pan-flavivirus and WNV-specific assays. Viral sequences in positive pools were characterized as WNV lineage 1 clade 1a and demonstrated 1-4% divergence. No flavivirus sequences other than WNV were detected in the mosquito pools. WNV infection rates in Oc. caspius and Cx. pipiens s.l. pools were 15.6% and 36.3%, respectively. Comparison of current and previously identified WNV sequences from Turkey revealed 94.00-96.34% similarity., Discussion: WNV RNA was identified for the first time in Cx. pipiens s.l. and Oc. caspius mosquitoes in Eastern Thrace, Turkey. Our findings indicate the circulation of WNV lineage 1 strains in potential vector species and provide an epidemiological link between WNV activity in mosquitoes and vertebrate infections.