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Real-time RT-PCR assay to detect Granada virus and the related Massilia and Arrabida phleboviruses

AuthorsDavó, Laura; Herrero, Laura ; Sánchez-Seco, María Paz; Labiod, Nuria; Roz, David; Gómez-Díaz, Elena ; Hernández, Lourdes; Figuerola, Jordi ; Vázquez, Ana
KeywordsReal-time RT-PCR
Granada virus
Massilia virus
Arrabida virus
Issue Date2020
PublisherBioMed Central
CitationParasites and Vectors, 13: 270 (2020)
AbstractBackground: Granada virus belongs to the genus Phlebovirus within the Naples serocomplex and was detected for the frst time in sand fies 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 specifc primers and one unique probe to detect Granada, Massilia and Arrabida viruses, without diferentiating between them, were designed targeting the conserved L-segment of their genome. Sensitivity was assessed using 10-fold serial dilutions of quantifed in vitro DNA samples. Specifcity was evaluated by testing diferent genomic RNA extracted from other representative phleboviruses. The new assay was used for virus detection in sand fies 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 fy samples studied from four diferent Balearic Islands, we detected one positive in the island of Cabrera. Conclusions: To our knowledge, the method described here is the frst 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 reli‑ able assay for the accurate diagnosis of human infections as well as for virus surveillance in vectors.
Publisher version (URL)https://doi.org/10.1186/s13071-020-04110-5
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