title: A rapid and specific real-time RT-PCR assay to identify Usutu virus in human plasma, serum, and cerebrospinal fluid.
abstract: BACKGROUND
  Usutu virus (USUV), a flavivirus that belongs to the Japanese encephalitis virus (JEV) family, has recently emerged as a human pathogen, necessitating new diagnostic tools.
  
  
  OBJECTIVE
  The development and assessment of a real-time RT-PCR assay to detect USUV in human samples.
  
  
  METHODS
  Based on USUV genomic sequences from GenBank, USUV-specific primers and probes that target the NS5 gene were designed. The sensitivity was evaluated in a 10-fold dilution series of plasmid that contained the amplicon and in a dilution series of a quantified human USUV isolate. The specificity was determined by testing various concentrations of related ArBo viruses, including flaviviruses and phleboviruses. Human RNAse P was also amplified in the assay. One hundred four human specimens from patients who suffered from viral meningoencephalitis were evaluated.
  
  
  RESULTS
  The real-time RT-PCR assay had a sensitivity of 50 genomic copies per reaction (corresponding to 2200 copies/ml) and 1 PFU/ml of USUV isolate. USUV isolates from Austria were identified with identical efficiency, and no ArBo viruses, other than USUV, were detected. USUV was also identified in 3 cerebrospinal fluid samples. All human samples were positive for RNAse P.
  
  
  CONCLUSIONS
  This PCR assay is recommended for all cases in which a rapid and clinically accurate diagnosis of human USUV infection is required.
authors.affiliation: Unit of Clinical Microbiology, Regional Reference Centre for Microbiological Emergencies (CRREM), St. Orsola Malpighi University Hospital, University of Bologna, Via G. Massarenti 9, 40138 Bologna, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Cavrini
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Della Pepa
authors.forename: Paolo
authors.initials: P
authors.lastname: Gaibani
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Pierro
authors.forename: Giada
authors.initials: G
authors.lastname: Rossini
authors.forename: Maria Paola
authors.initials: MP
authors.lastname: Landini
authors.forename: Vittorio
authors.initials: V
authors.lastname: Sambri
mesh_terms: D002555:Cerebrospinal Fluid / Q000821:virology*; D018349:Encephalitis Viruses, Japanese / Q000302:isolation & purification*; D004671:Encephalitis, Arbovirus / Q000175:diagnosis*; D018177:Flavivirus Infections / Q000175:diagnosis*; D006801:Humans; D015345:Oligonucleotide Probes / Q000235:genetics; D010949:Plasma / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D044967:Serum / Q000821:virology*; D017361:Viral Nonstructural Proteins / Q000235:genetics; D014773:Virology / Q000379:methods
chemical_list: D015345:Oligonucleotide Probes; D017361:Viral Nonstructural Proteins