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

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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.

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.

Section snippets

Background

Usutu virus (USUV) is a member of the genus Flavivirus (family Flaviviridae) first detected in Africa; it recently emerged causing infections in birds in Europe.1 In 2001, USUV caused mass mortalities in birds in Austria2 and then in Hungary,4 Switzerland,1 and Northern Italy.5, 6 The genomes of the Austrian USUV strain and the reference strain (SAAR-1776) were sequenced completely, sharing 97% nucleotide and 99% amino acid identity.7 In naturally infected birds, the virus does not have a

Objectives

Based on the medical need that has been raised by the recently reported detection of human USUV-caused neuroinvasive disease,8, 9 we developed a real-time RT-PCR assay to detect and distinguish USUV from other arboviruses, particularly from JEV members. This test was designed to identify viral isolates that have been circulating in Europe. The heminested RT-PCR assay that uses universal flavivirus primers10 is very sensitive, but it requires post-PCR sequencing to make a final and complete

Primers and probes

The nucleotide sequences of the Austrian and Hungarian USUV isolates (GenBank accession numbers: AY453411 and EF206350, respectively) were aligned to the most closely related flavivirus (JEV, MVEV, WNV) sequences in GenBank (Nucleotide accession numbers: NC_001437, NC_000943, NC_001563) using ClustalW (Version 2.0.12). A pair of primers and a probe were designed using Step One Primer Express 3.0. The specificity of the primers and probe for USUV was also assessed by BLAST (BLAST software is

Analytical performance

The real-time RT-PCR assay, designed for USUV detection, showed linear results for 7 logs of the USUV plasmid dilution series; the standard curve revealed an efficiency of 95.3% and a sensitivity of 50 copies/reaction (2200 copies/ml) of DNA template. Moreover the assay showed linear results for 6 logs of the USUV isolate, the standard curve revealed an efficiency of 94.6% and a sensitivity of 1 PFU/ml. Clinical samples that were positive for DENV-1, DENV-3, and WNV tested negative for USUV, as

Discussion

We designed and assessed the clinical applicability of a new real-time RT-PCR assay that was specific for USUV NS5 gene. This technique had high specificity; none of the potentially cross-reacting arboviruses was positive. This specificity raises the potential for the effective clinical application of our assay, because a commercially available, TMA-based NAT test that screens blood samples for WNV was recently reported to yield false-positive results during USUV viremia.15 The level of

Conflicts of interest

All the authors declared that there are no conflicts of interest.

Ethical approval

Not required.

Acknowledgments

This work was supported by the funds “LAB P3” (from Regione Emilia Romagna) and “RFO 2009” (from University of Bologna) to VS. We would like to thank Paolo Cordioli (IZS, Brescia, Italy) for the Austrian USUV isolate and ENIVD for providing some of the Arbovirus isolates.

References (16)

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