Construction of an internal standard used in RT nested PCR for Borna Disease Virus RNA detection in biological samples

Abstract

-The highly neurotropic Borna Disease Virus (BDV), which belongs to the Mononegavirales order -Bornaviridae family -is generally detected using the RT-nested-PCR. If false positive results (often caused by laboratory contaminations) can be avoided, some false negative results which are mostly due to inhibitory effects of some reaction components and/or to sample preparation errors, can occur. Thus, in order to control the RT-PCR sample, an RNA internal standard molecule named "mimic" was constructed with the same primer recognition sites as the viral nucleic acids, flanking a heterologous DNA fragment of distinct molecular weight. Because of their different sizes, the mimic and viral PCR products can be easily discriminated by agarose gel electrophoresis. The co-amplification of both BDV and mimic RNA was performed on infected cells and on biological tissues such as the brain and blood, commonly known to contain PCR inhibitor components. After mimic sensitivity studies were achieved (2.5 fg of "p40 RNA mimic" and 0.25 fg of "p24 RNA mimic"), the competitive amplification reaction between both BDV and mimic RNA was performed on these tissues. The results confirmed that nervous tissue has an inhibitory effect on RT-PCR, which supports the necessity of BDV detection by a higher sensitive method such as RT nested PCR. Moreover, these results confirmed the interest of an internal standard for BDV RNA detection in biological samples. Borna virus / PCR / internal standard / biological tissueRésumé -Construction d'un témoin interne de RT-PCR nichée pour la détection du génome du virus de la maladie de Borna dans des tissus biologiques. Le génome du virus de la maladie de Borna (« Borna disease Virus » -BDV), du fait des propriétés biologiques de ce virus, n'est généralement détectable que par la méthode RT-PCR nichée. Si les résultats « faux-positifs » peuvent être évités par des conditions de manipulation très strictes, les résultats « faux-négatifs » demeurent bien Vet. Res. 31 (2000) 565-572 565

Borna Disease Virus RNA in Immunocompromised Patients in Southwestern France

“…A classical extraction method was therefore chosen. Molecular techniques including an internal standard should certainly be preferred in future investigations (24,41). Questions about the best available method for achieving optimal detection of BDV RNA in blood and definitively eliminating false-negative results are still unanswered.…”

Borna disease: current knowledge and virus detection in France

“…The amplified BDV-products after the two successive PCR are 528 bp for the p40 gene and 391 bp for the p24 gene. In order to control RT-nested-PCR in each reaction sample and to avoid the use of a BDV-positive control reaction that could induce contamination, internal RNA standard molecules (named "mimics") have been produced [37]. These standards can be easily discriminated from the Borna fragments by agarose gel electrophoresis.…”

“…Apart from recent serological surveys that have revealed the presence of seropositive horses in France [7,20], only one recent study has searched for the BDV genome in France [37]. Even though no enzootic BDV has ever been reported in France, BDV is likely present in France, as it now is in several other countries.…”

“…The RT-nested-PCR described in 4.4.2 [37] was used to detect the viral genome in 171 animal (horse, fox, bovine, dog and sheep) brains and 25 horse blood samples. Some of the animals tested showed nervous disorders.…”

Evidence of Borna disease virus genome detection in French domestic animals and in foxes (Vulpes vulpes)

“…RNA detection by RT–nested-PCR was carried out using specific primers of the two BDV targets described by Sauder et al (1996) : the p40 gene, encoding the nucleoprotein, and the p24 gene, encoding the polymerase co-factor phosphoprotein. To avoid the risks of false-negative results, two internal standard molecules, named ‘mimic-p24’ and ‘mimic-p40’ were developed for RT–nested-PCR (Ballagi-Pordany & Belak, 1996 ; Legay et al , 2000 ). The internal standard molecules were produced by PCR (Ballagi-Pordany & Belak, 1996 ) from a cDNA fragment of the serotype 3 African horse sickness virus (an Orbivirus ) partial segment 2 (Sailleau et al , 2000 ) with specific ‘mimic primers’ (Legay et al , 2000 ).…”