AMPLIFICATION OF A FULL-LENGTH BORNA-DISEASE-VIRUS (BDV) CDNA FROM TOTAL RNA OF CELLS PERSISTENTLY INFECTED WITH BDV

We have developed a novel reverse transcriptase-polymerase chain react ion (RT-PCR) to amplify the full-length 8.9 kilobase (kbp) cDNA of the Borna disease virus (BDV) RNA genome from the total cellular RP;A of MDCK cells persistently infected with BDV (MDCK/BDV). Antigenomic BDV cDNA was reverse transcribed using a 53-mer oligonucleotide primer, co rresponding to the 5'-terminus of a putative 3'-leader sequence of the BDV RNA genome, for 2 hr at 42 C followed by 30 min at 55 C. PCR was performed in the presence of this 53-mer antigenomic primer and a 25-m er primer, corresponding to the 3'-terminus of the BDV antigenomic cDN A, by use of an rTth DNA polymerase with proof-reading activity. The a mplified full-length BDV cDNA was detected in as little as 20 ng of to tal cellular RNA of MDCK/BDV. This RT-PCR method should be a useful te chnique to study the molecular quasispecies of BDV.