Real-time RT-PCR Bornavirus type 1 (BDV-1) detection
Degree Final Project defended by Lucía Aroca Lara
September 18th, 2019
Bornavirus (BDV) is the causative agent of Borna disease (BD). Typically, it is described as a chronic, progressive, non-suppurative meningoencephalomyelitis which mainly affects horses and sheep, causing behavioural and neurological alterations. Currently, the possible zoonotic aspect of BD is very controversial.
BD diagnosis is very challenging. Clinical diagnosis is insufficient by itself as there are many differential diagnoses of pathogens which cause similar clinical signs in horses. So far, no diagnostic method has enough sensitivity and specificity to be used, on its own, in a BD clinical suspicion.
The aim of this study was to set up and validate a multiplex real time polymerase chain reaction for RNA (RT-PCR) for the simultaneous detection of two sequences corresponding to mammalian 1 Orthobornavirus (BDV-1) nucleoprotein N (p40) and phosphoprotein P (p24). It was carried out using the same primers and probes used in a study published in 2007 by Schindler et al. In addition, a screening was performed to detect a possible BDV-1 infection in 47 biological samples from 20 healthy horses (peripheral blood) and from 20 horses displaying neurological signs compatible with BD (peripheral blood, cerebrospinal fluid, and nasopharyngeal swabs) in order to detect a possible BDV-1 infection.
Although good efficiency and specificity were obtained in the real-time RT-PCR, there was amplification of p24 in late cycles in only 6 horse samples (2 with neurological signs and 4 healthy), but no amplification of p40. After running a 4% agarose gel electrophoresis of the amplification products from these 6 samples, no bands compatible with the amplification of the p24 fragment were observed.
In conclusion, the developed real-time RT-PCR is efficient and specific for BDV-1 detection in these samples. However, it is necessary to carry on assays in order to obtain similar results in the amplification of both genes (p40 and p24). The results obtained from the samples included in this study should be compared by means of a conventional nested PCR with a future wider sampling from healthy and horses displaying neurological signs.
