title: A rapid and specific real time RT-PCR assay for diagnosis of Toscana virus infection.
abstract: BACKGROUND
  To scan a virus (TOSV) belongs to the Phlebovirus genus within the Bunyaviridae family. TOSV is an arbovirus transmitted by sandflies. In Mediterranean countries, TOSV is one of the major viral pathogens involved in aseptic meningitis and meningoencephalitis.
  
  
  OBJECTIVE
  Development and assessment of a new sensitive and specific real-time RT-PCR assay for TOSV diagnosis.
  
  
  METHODS
  TOSV-specific primers and probe targeting the S-segment of the genome were designed, based on recent TOSV sequences available in public databases. Sensitivity was assessed using 10-fold serial dilutions of a RNA transcript and serial dilutions of TOSV strains isolated from infected human beings. Specificity was determined by testing RNA extracts from closely related Phleboviruses. The assay was then used for TOSV infection diagnosis in 971 clinical samples and for TOSV detection in 2000 sandflies.
  
  
  RESULTS
  The real-time RT-PCR assay exhibited a sensitivity of under 257 copies per reaction for the RNA transcripts and 0.0056 and 0.014 TCID50 of Italian and Spanish TOSV genotypes per reaction, respectively. No other close Phleboviruses were detected. TOSV was identified in 17 clinical samples and in 3 sandflies.
  
  
  CONCLUSIONS
  The assay described is a rapid, robust and reliable real-time RT-PCR test for accurate diagnosis of human TOSV infection as well as for the surveillance of TOSV in vector populations.
authors: UMR 7268 ADES, Aix-Marseille Université/EFS/CNRS, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France. Electronic address: nadege.brisbarre@gmail.com.
authors: Nadège
authors: N
authors: Brisbarre
authors: CNR Arbovirus IRBA (Institut de Recherche Biomédicale des Armées), 111 Avenue de la Corse BP40026, 13568 Marseille Cedex 02, France. Electronic address: sebastien.plumet@intradef.gouv.fr.
authors: Sébastien
authors: S
authors: Plumet
authors: Unité d'entomologie Médicale, IRBA (Institut de recherche Biomédicale des Armées), 111 Avenue de la Corse BP40026, 13568 Marseille Cedex 02, France. Electronic address: lautard.christelle@gmail.com.
authors: Christelle
authors: C
authors: Cotteaux-Lautard
authors: CNR Arbovirus IRBA (Institut de Recherche Biomédicale des Armées), 111 Avenue de la Corse BP40026, 13568 Marseille Cedex 02, France. Electronic address: sebastien.emonet@irba.fr.
authors: Sébastien F
authors: SF
authors: Emonet
authors: Unité d'entomologie Médicale, IRBA (Institut de recherche Biomédicale des Armées), 111 Avenue de la Corse BP40026, 13568 Marseille Cedex 02, France. Electronic address: frederic.pages@ars.sante.fr.
authors: Frédéric
authors: F
authors: Pages
authors: CNR Arbovirus IRBA (Institut de Recherche Biomédicale des Armées), 111 Avenue de la Corse BP40026, 13568 Marseille Cedex 02, France. Electronic address: isabelle.leparcgoffart@gmail.com.
authors: Isabelle
authors: I
authors: Leparc-Goffart
mesh_terms: D002044:Bunyaviridae Infections / Q000175:diagnosis* / Q000821:virology; D005260:Female; D006801:Humans; D007558:Italy; D008297:Male; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D029301:Sandfly fever Naples virus / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D013030:Spain; D013997:Time Factors
keywords: Arbovirus; Bunyaviridae; Diagnosis; Real-time RT-PCR; Toscana virus