A rapid and specific real-time RT-PCR assay to identify Usutu virus in human plasma, serum, and cerebrospinal fluid

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 detectUSUVinhumansamples. Study design: 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