Molecular double-check strategy for the identification and characterization of European Lyssaviruses

Melina Fischer,Conrad M Freuling,Thomas Müller,Anne Wegelt,Engbert A Kooi,Thomas B Rasmussen,Katja Voller,Denise A Marston,Anthony R Fooks,Martin Beer,Bernd Hoffmann

doi:10.1016/j.jviromet.2014.03.014

Melina Fischer, Conrad M Freuling + Show 9 more

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https://doi.org/10.1016/j.jviromet.2014.03.014

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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.

Highlights

Rabies has been known since antiquity, it still represents one of the most important zoonotic diseases with an immense public health impact, especially in developing countries (Knobel et al, 2005; Anderson and Shwiff, 2013)
Rhabdovirus isolates of Vesicular stomatitis New Jersey virus (VSV NJ), Vesicular stomatitis Indiana virus (VSV Ind), Bovine ephemeral fever virus (BEFV) and Viral hemorrhagic septicemia virus (VHSV) were analyzed for specificity testing using both pan-lyssavirus RT-qPCRs
Numerous molecular techniques have been developed for lyssavirus detection, the diversity of this genus implies certain limitations, especially concerning their diagnostic range (Fooks et al, 2009; Coertse et al, 2010; Hoffmann et al, 2010)

Summary

Introduction

Rabies has been known since antiquity, it still represents one of the most important zoonotic diseases with an immense public health impact, especially in developing countries (Knobel et al, 2005; Anderson and Shwiff, 2013). The causative agents of this fatal encephalitis are lyssaviruses (order Mononegavirales, family Rhabdoviridae), with the prototype species being Rabies virus (RABV). The two most recently discovered lyssaviruses, Bokeloh bat lyssavirus (BBLV) and Ikoma virus (IKOV) (Freuling et al, 2011; Marston et al, 2012), are approved as new lyssavirus species and are awaiting ratification by the International Committee on Taxonomy of Viruses. Bats appear to be the original reservoir for lyssaviruses, and many of those bat-associated viruses have caused human fatalities worldwide (Johnson et al, 2010; Banyard et al, 2011). The vast majority of human exposures and fatalities are related to RABV, which is transmitted by dog bites (WHO, 2013)

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