title: Development of a Usutu virus specific real-time reverse transcription PCR assay based on sequenced strains from Africa and Europe.
abstract: Usutu virus (USUV) has been isolated in several African and European countries mainly from mosquitoes and birds. However, previous benign and two recent severe cases of human infections point out the need of a tool for the identification of USUV in human samples. A published real-time reverse transcription (RT) PCR assay for the detection of USUV in human blood or cerebrospinal fluid does not take into account the genetic variability of USUV in different geographic regions. Therefore, this article presents a quantitative real-time RT-PCR assay based on sequences from Europe and Africa. Primers and probe were designed in conserved regions among USUV strains that differed from closely related flaviviruses. The specificity of the assay was investigated by testing 16 other flaviviruses circulating in Africa. The sensitivity was determined by testing serial dilutions of virus and RNA standard. Intra- and inter-assay coefficients of variation were evaluated by 10 reactions in a same and in different assays, respectively. The assay provides high analytical specificity for USUV and detection limits of 1.2pfu/reaction for virus dilutions in L-15 medium or human serum and 60 copies/reaction for the RNA standard. The assay needs to be evaluated in a clinical context and integrated in standard diagnosis of flaviviral diseases.
authors.affiliation: Unité des arbovirus et virus de fièvres hémorragiques, Institut Pasteur de Dakar, 34 Avenue Pasteur, Dakar, Senegal; Université Cheikh Anta Diop Dakar, 24 Avenue Cheikh Anta Diop, Dakar, Senegal; University Vienna, Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria. Electronic address: birgitnikolay@gmail.com.
authors.forename: B
authors.initials: B
authors.lastname: Nikolay
authors.affiliation: Department of Virology, University Medical Center Goettingen, Kreuzbergring 57, D-37075 Goettingen, Germany. Electronic address: mweidma@gwdg.de.
authors.forename: M
authors.initials: M
authors.lastname: Weidmann
authors.affiliation: CNRS UMR 8122, Institut Gustave Roussy, Villejuif 94805, France. Electronic address: dupresso@igr.fr.
authors.forename: A
authors.initials: A
authors.lastname: Dupressoir
authors.affiliation: Unité des arbovirus et virus de fièvres hémorragiques, Institut Pasteur de Dakar, 34 Avenue Pasteur, Dakar, Senegal. Electronic address: ofaye@pasteur.sn.
authors.forename: O
authors.initials: O
authors.lastname: Faye
authors.affiliation: University Vienna, Dr. Bohr-Gasse 9/3, A-1030 Vienna, Austria. Electronic address: cheikh.boye@ucad.edu.sn.
authors.forename: C S
authors.initials: CS
authors.lastname: Boye
authors.affiliation: Unité d'entomologie médicale, Institut Pasteur de Dakar, Senegal.
authors.forename: M
authors.initials: M
authors.lastname: Diallo
authors.affiliation: Unité des arbovirus et virus de fièvres hémorragiques, Institut Pasteur de Dakar, 34 Avenue Pasteur, Dakar, Senegal. Electronic address: asall@pasteur.sn.
authors.forename: A A
authors.initials: AA
authors.lastname: Sall
mesh_terms: D000349:Africa; D017931:DNA Primers / Q000235:genetics; D018349:Encephalitis Viruses, Japanese / Q000235:genetics / Q000302:isolation & purification*; D004671:Encephalitis, Arbovirus / Q000175:diagnosis* / Q000821:virology; D005060:Europe; D018177:Flavivirus Infections / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers
keywords: CV; Diagnosis; Flavivirus; R; Real-time RT-PCR; Standard RNA; USUV; Usutu virus; coefficient of variation; correlation coefficient