Development of a quantitative real-time RT-PCR assay with internal control for the laboratory detection of tick borne encephalitis virus (TBEV) RNA

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Abstract

Background: Tick borne encephalitis virus (TBEV), is a human flavivirus causing tick borne encephalitis (TBE), a viral infection of the central nervous system endemic in Europe and Asia. Objectives: To develop a reverse transcription polymerase chain reaction (RT-PCR) assay based on quantitative real-time RT-PCR technology (TaqMan) for detection and quantification of TBEV RNA. The test includes an internal control (IC) to avoid false negative results. Study design: The system was established and validated using wild-type (WT) non-infectious synthetic RNA representing a fragment of the 3′ non-coding region of the TBEV genome. In addition, synthetic RNA differing from the WT synthetic RNA by a unique probe binding region was used as IC to monitor the overall efficiency of the RT-PCR. Results: The analytical sensitivity of the assay was at least ten copies of the TBEV synthetic transcript in presence of 50 copies of the IC. Successful amplification was obtained for different strains within the TBEV complex (Hypr, Hochosterwitz, Laibach, Elsass=Alsace, ZZ9, Wladiwostok). Among 14 serum and 21 cerebrospinal fluid (CSF) samples obtained from 28 patients with clinical suspicion of TBEV 1 CSF sample tested positive for TBEV RNA. In addition, no TBEV RNA could be detected in blood samples obtained from three vaccinated people 1 and 3 days post-vaccination. Thus indicating that a positive result is unlikely to be caused by recent vaccination. Conclusions: A quantitative, highly sensitive and specific real-time RT-PCR assay has been developed for the detection of TBEV RNA. Inclusion of an IC is important to monitor the possible occurrence of false-negative results caused by the presence of inhibitory factors. This assay should be an important asset for the routine laboratory detection of TBEV RNA.

Introduction

Tick borne encephalitis virus (TBEV) belongs to the family Flaviviridae and causes tick borne encephalitis (TBE) in humans, a disease mainly involving the central nervous system. Viruses in the family Flaviviridae include the etiological agents of Dengue fever, Yellow fever, Japanese encephalitis, St. Louis encephalitis and hepatitis C to name a few. As for all flaviviruses, the TBEV genome consists of a linear positive-stranded RNA molecule of approximately 10.5 kb in length (Wallner et al., 1995, Mandl et al., 1991). A single long open reading frame flanked by two short non-coding sequences encodes for all viral proteins (Wallner et al., 1996).

There are two subtypes of TBEV, the Western and the Far Eastern subtype. The Western subtype is transmitted by the tick Ixodes ricinus and the Far Eastern subtype by the tick Ixodes persulcatus. TBE is endemic to northern, central and eastern Europe, Russia and the Far East (Dumpis et al., 1999).

An increase in the reported number of cases together with the severity of the symptoms caused by infection with TBEV compels for the development of appropriate diagnostic methods. So far, diagnostic evidence and epidemiological studies rely usually on serological testing (Dumpis et al., 1999). However, correct interpretation of serological tests is often complicated by a number of factors such as cross reactions with antibodies directed against other virus species, past infections with TBEV or recent vaccination. Furthermore, and most importantly, antibody assays may be negative in the early phase of the disease when clinical symptoms are manifest (Dumpis et al., 1999). This may be caused by a delayed immune response. These various diagnostic difficulties have prompted us to establish a real-time reverse transcription polymerase chain reaction (RT-PCR) assay based on TaqMan chemistry for the laboratory detection of TBEV RNA. The principle of real time RT-PCR consists in a reverse transcription step, followed by PCR amplification. During PCR the TaqMan probe is cleaved by the 5′–3′ nuclease activity of the TaqDNA polymerase. The TaqMan probe contains a fluorescent reporter dye at the 5′-end and a fluorescent quencher dye at the 3′-end. Cleavage of the reporter dye during PCR results in increased fluorescence, which is directly proportional to the accumulation of PCR products. Whereas RT-PCR assays have been previously described to successfully detect TBEV RNA in ticks and patient samples (Ramelow et al., 1993, Whitby et al., 1993, Suess et al., 1997, Schrader and Suess, 1999, Wicki et al., 2000), we developed a test presenting with additional features such as quantification of viral RNA, elimination of post-PCR processing steps, the use of a closed one-tube system for both the reverse-transcription and the amplification steps and the inclusion of an internal control (IC) into each reaction.

The technical characteristics of the quantitative real-time RT-PCR assay for detection and quantification of TBEV RNA are described in the present study. In addition, preliminary results will illustrate its routine applicability to detect TBEV RNA isolated from clinical samples.

Section snippets

Viral strains

The following strains of the European subtype of TBEV were obtained from Professor F.X. Heinz (Institute of Virology, University Vienna): ZZ9 isolated from a tick in Tyrol, Austria, in 1985, (Guirakhoo et al., 1987), Hochosterwitz, isolated from a tick in Carinthia, Austria, in 1971 (Heinz et al., 1981), Hypr, isolated from human blood in Czechoslovakia in 1953 (Suess et al., 1997), Laibach I isolated in Slovenia in 1993, Elsass=Alsace isolated in France in 1990. For the Far Eastern subtype we

Selection of optimal concentrations for primers and probes for the TBEV RT-PCR

Various concentrations of primers (50/300/900 nM) and of the TaqMan probe (100/200/300 nM) were tested using 10 000 copies of the TBE-WT transcript as template. Following RT-PCR, the concentrations of primers and probe giving the highest fluorescence and the lowest threshold cycle (Ct) were selected as follows: 50 nM forward primer F-TBE 1, 300 nM reverse primer R-TBE 1 and 200 nM TBE-Probe-WT. In order to avoid background signals and interference with the wild-type probe, the concentration of

Discussion

The importance of tick borne viral encephalitis as a seasonal disease has been emphasised in various recent publications (Ruef, 2000, Schwanda et al., 2000). The incidence of TBE infections is increasing and endemic regions are constantly expanding. In Switzerland 108 cases have been registrated by the Swiss Federal Office of Public Health (http://www.bag.admin.ch) for the year 2001. TBE, which may occur from Spring to late Autumn, should be considered in the differential diagnosis of

Acknowledgements

Financial support for this project was provided by the AC-Laboratorium Spiez, Switzerland (Dr. M. Schütz). We thank Professor G. Siegl (St. Gallen, Switzerland) for critical reading of the manuscript and Dr. M. Weitz (St. Gallen, Switzerland) for his continuous support. Furthermore, we thank Professor F.X. Heinz (Vienna, Austria) and Dr. C. Moll (Münsterlingen, Switzerland) for providing us with specimens or specific information.

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