Development of a Usutu virus specific real-time reverse transcription PCR assay based on sequenced strains from Africa and Europe

• Published in: Journal of virological methods 2014-03, Vol.197, p.51-54
• Main Authors: Nikolay, B., Weidmann, M., Dupressoir, A., Faye, O., Boye, C.S., Diallo, M., Sall, A.A.
• Format: Article
• Language: English
• Subjects: Africa; Diagnosis; DNA Primers - genetics; Encephalitis Viruses, Japanese - genetics; Encephalitis Viruses, Japanese - isolation & purification; Encephalitis, Arbovirus - diagnosis; Encephalitis, Arbovirus - virology; Europe; Flavivirus; Flavivirus Infections - diagnosis; Flavivirus Infections - virology; Humans; Real-Time Polymerase Chain Reaction - methods; Real-time RT-PCR; Reverse Transcriptase Polymerase Chain Reaction - methods; Sensitivity and Specificity; Standard RNA; Usutu virus
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2013.08.039

•We developed a USUV specific 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 assay was able to detect all five tested USUV isolates from Africa.•The assay provides high analytical specificity for USUV tested with 16 other flaviviruses.•Detection limits were 1.2pfu/reaction for virus dilutions and 60 copies/reaction for the RNA standard. 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.