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 reaction (RT-PCR) to amplify the full-length 8.9 kilobase (kbp) cDNA of the Borna disease virus (BDV) RNA genome from the total cellular RNA of MDCK cells persistently infected with BDV (MDCK/BDV). Antigenomic BDV cDNA was reverse transcribed using a 53-mer oligonucleotide primer, corresponding 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-mer primer, corresponding to the 3'-terminus of the BDV antigenomic cDNA, by use of an rTth DNA polymerase with proof-reading activity. The amplified full-length BDV cDNA was detected in as little as 20 ng of total cellular RNA of MDCK/BDV. This RT-PCR method should be a useful technique to study the molecular quasispecies of BDV.
Shoya
Y
Y
Institute of Immunological Science, Hokkaido University, Japan.
Kobayashi
T
T
Koda
T
T
Lai
P K
PK
Tanaka
H
H
Koyama
T
T
Ikuta
K
K
Kakinuma
M
M
Kishi
M
M
D000818:Animals; D001891:Borna disease virus / Q000235:genetics*; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D004285:Dogs; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics*; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral