Reverse transcription real-time PCR assays for detection and quantification of Borna disease virus in diseased hosts

Abstract

Borna disease is a severe, immunopathological disorder of the central nervous system caused by the infection with the Borna disease virus (BDV). The detection of BDV in diseased animals, mainly sheep and horses, is achieved by histological, immunohistochemical and serological approaches and/or PCR-based technologies. In the present study, reverse transcription, real-time PCR assays were established for the detection of BDV in the brain tissue from sheep and horses, using loci for the p40 (nucleoprotein) and the p24 (phosphoprotein) genes. The PCRs were equally specific and sensitive, detecting 10 target molecules per reaction and one BDV-infected cell among 10(6) non-infected cells. In tissues from BDV-diseased sheep and horses, the p24 target was detected at higher abundance than for p40. Therefore, the p24 test is suggested to be of higher value in the diagnostic laboratory. However, both assays should be useful for addressing questions in pathogenesis and for detecting BDV reservoirs in endemic areas.

Fig. 1

Sensitivity of detection of BDV-infected MDCK cells. BDV-infected MDCK cells were diluted in 10-fold dilution steps and complemented with non-infected cells to obtain a uniform number of 10⁶ cells before RNA extraction for reverse transcription, real-time PCR. The threshold for both primers was 0.00004 (dashed line), representing the normalized and calibrated value from non-infected MDCK cells (see materials and methods). White bars: amplification with p40 primers; gray bars: with p24 primers; x-axis: number of infected MDCK cells; y-axis: relative BDV copy number.

Fig. 2

Relative amounts of BDV templates in various brain regions of BDV-diseased sheep. Extracts from triplicate samples of different brain sites of three different BDV-diseased sheep (S03-0057, S02-1736 and S99-1356) were subjected to reverse transcription and real-time PCR. Box plots are shown, representing maximal, average, median, and minimal numbers of BDV-specific template detected. The signals of the BDV-specific amplifications are expressed as arbitrary units (y-axis) relative to the 18S rRNA signal, which normalizes the total number of cells in the assay. Amplifications for p40 and p24 were done simultaneously, using the same volume of the same template. The threshold for both primers was 0.005. x-axis: brain sites and PCR system; y-axis: relative BDV copy number.

Fig. 3

Relative amounts of BDV templates in various brain regions of BDV-diseased horses. Extracts from triplicate samples of cortex and hippocampus of three different BDV-diseased horses (S00-0913, S99-0598 and S96-0868) as well as lobus piriformis from two horses and cerebellum from one horse were subjected to reverse transcription and real-time PCR. Box plots are shown, representing maximal, average, median, and minimal numbers of BDV-specific template detected. The signals of the BDV-specific amplifications are expressed as arbitrary units (y-axis) relative to the 18S rRNA signal, which normalizes the total number of cells in the assay. Amplifications for p40 and p24 were done simultaneously, using the same volume of the same template. The threshold for both primers was 0.0004. x-axis: brain sites and PCR system; y-axis: relative BDV copy number.