Davo L. auteur aut IRD Herrero L. auteur aut IRD Sanchez-Seco M. P. auteur aut IRD Labiod N. auteur aut IRD Roiz David auteur aut IRD Gomez-Diaz E. auteur aut IRD Hernandez L. auteur aut IRD Figuerola J. auteur aut IRD Vazquez A. auteur aut IRD text journalArticle eng text/pdf reformatted digital access Background Granada virus belongs to the genus Phlebovirus within the Naples serocomplex and was detected for the first time in sand flies from Spain in 2003. Seroprevalence studies have revealed that Granada virus may infect humans with most cases being asymptomatic. Moreover, recent studies in vector samples revealed that the related Massilia and Arrabida phleboviruses could be also circulating in Spain. The objective of this study was to develop and assess a new sensitive real-time RT-PCR assay for Granada virus diagnosis able to detect the related phleboviruses Massilia and Arrabida. Methods Two specific primers and one unique probe to detect Granada, Massilia and Arrabida viruses, without differentiating between them, were designed targeting the conserved L-segment of their genome. Sensitivity was assessed using 10-fold serial dilutions of quantified in vitro DNA samples. Specificity was evaluated by testing different genomic RNA extracted from other representative phleboviruses. The new assay was used for virus detection in sand flies collected in 2012 from the Balearic Archipelago, a touristic hotspot in the Mediterranean. Results The real-time RT-PCR assay exhibited a sensitivity per reaction of 19 copies for Granada and Arrabida, and 16 copies for Massilia. No other related phleboviruses were detected. From the 37 pools of sand fly samples studied from four different Balearic Islands, we detected one positive in the island of Cabrera. Conclusions To our knowledge, the method described here is the first real-time RT-PCR designed to detect Granada virus and the related Massilia and Arrabida phleboviruses. The study demonstrated that this is a rapid, robust and reliable assay for the accurate diagnosis of human infections as well as for virus surveillance in vectors. specialized Real-time RT-PCR Granada virus Massilia virus Arrabida virus Diagnosis Surveillance 052 020 13 1 art. 270 [7 p.] 2020 1756-3305 https://www.documentation.ird.fr/hor/fdi:010078165 10.1186/s13071-020-04110-5 1756-3305 [F B010078165] https://www.documentation.ird.fr/hor/fdi:010078165 https://horizon.documentation.ird.fr/exl-doc/pleins_textes/divers20-06/010078165.pdf IRD - Base Horizon / Pleins textes 2020-07-02 2020-07-03 fdi:010078165 fre