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Phlebotomine sandflies are vectors of a large variety of arboviruses, mostly in the genus Phlebovirus. Many were discovered during the last decade, but were only partially sequenced because of extremely high genetic diversity. Next-Generation sequencing (NGS) is a key tool to achieve rapid sequencing of these viruses. Here we present the results obtained with a custom-made automated platform for high throughput processing (HTP) of field-trapped sandflies. The aim is to present an integrated approach to discover new/existing viruses vectored by sandflies, to characterize these viruses by complete genome sequencing, and to study their potential public health impact for humans and non human vertebrates. Sandflies were collected in France, Tunisia, Algeria, Turkey and Iran. They were processed on a custom-made automated HTP platform. RT-PCR assays targeting conserved regions within the genus Phlebovirus were performed both directly on sandfly extracted RNA and on supernatant of Vero cells, and further sequenced. Sandflies and cell culture that were PCR-positive, as well as supernatant of flasks with cytopathic effect were sequenced using a de novo protocol onto a Ion Torrent PGM sequencer. A total of 29,664 sandflies were collected and processed onto the platform. Origin and number of specimens are as follows: France (n = 3,087), Tunisia (n = 8,206), Algeria (n = 1,283), Turkey (n = 12,000), Iran (n = 5,089). We isolated 9 virus strains in France (2 Toscana, 7 Massilia), 10 strains in Tunisia (2 Toscana, 5 Punique, 3 new viruses still unnamed), 1 strain in Algeria (1 Toscana), 2 strains in Turkey (new phlebovirus, provisionally named Damyeri), 1 strain in Iran (new phlebovirus, provisionally named Dashli). Analysis using complete gene sequences allowed to produce full protein phylograms for isolated virus strains. Seroprevalence studies were performed to define whether these viruses infect humans and non human vertebrates in regions where sandflies were trapped or not. Seroprevalence results will be presented for France and Tunisia. A total of 23 strains were isolated, 6 of which correspond to new sandfly-borne phleboviruses that have been completely sequenced through NGS. Screening field-trapped sandflies is suitable to inventory recognized and new viruses. Combining cell culture for isolation and NGS for sequencing is cost-effective and adapted to process high quantities of insects. Serological tools are a rapid and easy method to address potential public health impact which must be confirmed by molecular diagnostic derived from complete sequence data obtained through NGS.

Background Cutaneous leishmaniasis is endemic to the Pacific coast of Ecuador, and Nyssomyia trapidoi is considered to be its main vector. Dujardin et al. [1] recorded some differences in body pigmentation and isoenzymatic profiles in sympatric populations of Ny. trapidoi from the Pacific coast of Ecuador and suggested the existence of two cryptic species. Methods Entomological collections were performed in November 2008 and March 2011 in the locality of Paraíso Escondido using CDC miniature light traps and human bait. Morphological, isoenzymatical and molecular (sequencing of cytochome b and cytochrome c oxidase 1 of the mitochondrial DNA) analyses, such as detection of Leishmania DNA and phlebovirus RNA in some females, were performed. Results Neighbor-joining trees from mitochondrial sequences grouped all of Ecuadorian Ny. trapidoi (including the two color variants) in one cluster, except for two specimens which clustered separately in both genes. Isoenzymatic characterization confirmed that the color variants belong to the same population. Additionally, 11.5% of females were found by PCR to contain Endotrypanum monterogeii kinetoplastid DNA. All pools of Ny. trapidoi were negative for phlebovirus RNA. Conclusion Analysis of mitochondrial gene sequences and isoenzymes was unable to support the existence of two sibling species within Ny. trapidoi, which is a probable vector of Endotrypanum monterogeii.