title: Molecular double-check strategy for the identification and characterization of European Lyssaviruses.
abstract: The "gold standard" for post-mortem rabies diagnosis is the direct fluorescent antibody test (FAT). However, in the case of ante-mortem non-neural sample material or decomposed tissues, the FAT reaches its limit, and the use of molecular techniques can be advantageous. In this study, we developed and validated a reverse transcription PCR cascade protocol feasible for the classification of samples, even those for which there is no epidemiological background knowledge. This study emphasises on the most relevant European lyssaviruses. In a first step, two independent N- and L-gene based pan-lyssavirus intercalating dye assays are performed in a double-check application to increase the method's diagnostic safety. For the second step, characterization of the lyssavirus positive samples via two independent multiplex PCR-systems was performed. Both assays were probe-based, species-specific multiplex PCR-systems for Rabies virus, European bat lyssavirus type 1 and 2 as well as Bokeloh bat lyssavirus. All assays were validated successfully with a comprehensive panel of lyssavirus positive samples, as well as negative material from various host species. This double-check strategy allows for both safe and sensitive screening, detection and characterization of all lyssavirus species of humans and animals, as well as the rapid identification of currently unknown lyssaviruses in bats in Europe.
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, D-17493 Greifswald-Insel Riems, Germany.
authors.forename: Melina
authors.initials: M
authors.lastname: Fischer
authors.affiliation: Institute of Molecular Biology, Friedrich-Loeffler-Institut, Südufer 10, D-17493 Greifswald-Insel Riems, Germany.
authors.forename: Conrad M
authors.initials: CM
authors.lastname: Freuling
authors.affiliation: Institute of Molecular Biology, Friedrich-Loeffler-Institut, Südufer 10, D-17493 Greifswald-Insel Riems, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Müller
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, D-17493 Greifswald-Insel Riems, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Wegelt
authors.affiliation: Central Veterinary Institute of Wageningen UR, Houtribweg 39, NL-8221 RA Lelystad, The Netherlands.
authors.forename: Engbert A
authors.initials: EA
authors.lastname: Kooi
authors.affiliation: DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Rasmussen
authors.affiliation: Animal Health & Veterinary Laboratories Agency (AHVLA, Weybridge), New Haw, Addlestone, Surrey KT15 3NB, United Kingdom.
authors.forename: Katja
authors.initials: K
authors.lastname: Voller
authors.affiliation: Animal Health & Veterinary Laboratories Agency (AHVLA, Weybridge), New Haw, Addlestone, Surrey KT15 3NB, United Kingdom.
authors.forename: Denise A
authors.initials: DA
authors.lastname: Marston
authors.affiliation: Animal Health & Veterinary Laboratories Agency (AHVLA, Weybridge), New Haw, Addlestone, Surrey KT15 3NB, United Kingdom; Department of Clinical Infection, Microbiology and Immunology, Institute of Infection and Global Health, University of Liverpool, Liverpool, Merseyside L69 7BE, United Kingdom.
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Fooks
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, D-17493 Greifswald-Insel Riems, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Beer
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, D-17493 Greifswald-Insel Riems, Germany. Electronic address: bernd.hoffmann@fli.bund.de.
authors.forename: Bernd
authors.initials: B
authors.lastname: Hoffmann
mesh_terms: D000465:Algorithms; D000818:Animals; D002685:Chiroptera; D005060:Europe; D006801:Humans; D018114:Lyssavirus / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D011818:Rabies / Q000175:diagnosis* / Q000662:veterinary* / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
keywords: Lyssavirus; Molecular diagnostics; Rabies; Real-time RT-PCR