Real-time RT-PCR assay to detect Granada virus and the related Massilia and Arrabida phleboviruses.
Arrabida virus
Diagnosis
Granada virus
Massilia virus
Real-time RT-PCR
Surveillance
Journal
Parasites & vectors
ISSN: 1756-3305
Titre abrégé: Parasit Vectors
Pays: England
ID NLM: 101462774
Informations de publication
Date de publication:
29 May 2020
29 May 2020
Historique:
received:
11
11
2019
accepted:
27
04
2020
entrez:
31
5
2020
pubmed:
31
5
2020
medline:
2
2
2021
Statut:
epublish
Résumé
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. 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. 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. 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.
Sections du résumé
BACKGROUND
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
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
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
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.
Identifiants
pubmed: 32471505
doi: 10.1186/s13071-020-04110-5
pii: 10.1186/s13071-020-04110-5
pmc: PMC7257231
doi:
Substances chimiques
DNA Primers
0
RNA, Viral
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
270Subventions
Organisme : Instituto de Salud Carlos III
ID : CP13/00114
Organisme : Instituto de Salud Carlos III
ID : PI14CIII/00014
Organisme : European Commission
ID : ERG-PARIS-276838
Organisme : Ministerio de Economía y Competitividad
ID : CGL2009-11445
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