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.

Development and validation of sensitive real-time RT-PCR assay for broad detection of rabies virus

“…Although in the comparative analysis, the L gene-based assay offered better performances than the N-gene assay regarding sensitivity and predictive values, it seems reasonable to perform a double-check strategy on tested samples, using both assays in order to increase reliability during use in routine rabies identification or epidemiological surveys as previously reported for others lyssaviruses (46,31). Moreover, high AUC value found in ROC analysis predicted that both assays are useful for accurate detection of RABV in positive samples.…”

“…Despite a higher sensitivity, hemi-nested RT-PCR presents some disadvantages in terms of workload, risk of contamination, and time. Thus, several real time molecular tests targeting the N or L gene have been developed to complement conventional diagnosis of rabies and rabies-related viruses (10,21,25,26,27,28,29,30). However, none of them have been validated against African strains from a large diversity of geographical origins, except a recent study (31).…”

Lagos bat virus transmission in an Eidolon helvum bat colony, Ghana

“…The samples were subject to several studies giving a unique inside into bat virus dynamics Drexler et al, 2012Drexler et al, , 2013Freidl et al, 2015). For lyssavirus surveillance brain samples were screened using a newly developed generic real-time RT-PCR assay (Fischer et al, 2014). Viral RNA was isolated with QIAamp RNeasy kit (Qiagen) according to manufacturer's instruction, and pools of five RNA samples were tested as described (Fischer et al, 2014).…”

“…For lyssavirus surveillance brain samples were screened using a newly developed generic real-time RT-PCR assay (Fischer et al, 2014). Viral RNA was isolated with QIAamp RNeasy kit (Qiagen) according to manufacturer's instruction, and pools of five RNA samples were tested as described (Fischer et al, 2014). Two samples yielded positive reactions.…”

>b<Estudo da distribuição do vírus da raiva (RABV) em amostras de sistema nervoso central e glândulas salivares de equinos naturalmente infectados>/b<

“…Devido a isso, essa técnica não deve ser utilizada em material encefálico fresco post-mortem provenientes dos animais e humanos suspeitos de raiva. No entanto, a RT-PCR pode ser utilizada em amostras clínicas coletadas de humanos suspeitos de raiva (como saliva, liquor, folículo piloso da nuca) para diagnóstico antemortem(DACHEUX et al, 2010;FISCHER et al, 2014).Após esse procedimento gerar uma amplificação positiva, a caracterização da sequência do produto de RT-PCR por meio de análises filogenéticas pode elucidar o tipo de variante viral e área geográfica aonde o vírus circula(FOOKS et al, 2009). Adicionalmente, a RT-PCR possui a vantagem de ser utilizado para detecção do ácido nucleico em amostras autolisadas, situações nas quais os testes convencionais podem ser ineficazes(DAVID et al, 2002).Outra situação na qual o desempenho da IFD pode estar diminuído, é quando há baixa carga viral, assim as diferentes provas moleculares também podem ser utilizadas como técnicas confirmatórias.…”