Development of infectious transcripts and genome manipulation of black queen-cell virus of honey bees
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Date
2002
Authors
Journal Title
Journal ISSN
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Publisher
Microbiology Society
Abstract
The South African isolate of Black queen-cell virus (BQCV), a honey bee virus, was previously found
to have an 8550 nucleotide genome excluding the poly(A) tail. Its genome contained two ORFs, a
5«-proximal ORF encoding a putative replicase protein and a 3«-proximal ORF encoding a capsid
polyprotein. Long reverse transcription (RT)–PCR was used to produce infectious transcripts for
BQCV and to manipulate its genome. Primers were designed for the amplification of the complete
genome, the in vitro transcription of infectious RNA and PCR-directed mutagenesis. An 18-mer
antisense primer was designed for RT to produce full-length single-stranded cDNA (ss cDNA).
Unpurified ss cDNA from the RT reaction mixture was used directly as a template to amplify the full
genome by long high-fidelity PCR. The SP6 promoter sequence was introduced into the sense
primer to transcribe RNA directly from the amplicon. RNA was transcribed in vitro with and without
the presence of a cap analogue and injected directly into bee pupae, which were then incubated for
8 days. In vitro transcripts were infectious but the presence of a cap analogue did not increase the
amount of virus recovered.
Description
Keywords
Biotechnology, Honey bee virus, South Africa, Mutant virus, Virology
Citation
Benjeddou, M. et al. (2002). Development of infectious transcripts and genome manipulation of black queen-cell virus of honey bees. Journal of General Virology, 83(12) , 3139-3146. 10.1099/0022-1317-83-12-3139