Development of infectious transcripts and genome manipulation of Black queen-cell virus of honey bees

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. A single base mutation abolishing an EcoRI restriction site was introduced by fusion-PCR, to distinguish viral particles recovered from infectious transcripts from wild-type virus (wtBQCV). Mutant virus (mutBQCV) and wtBQCV were indistinguishable by electron microscopy and Western blot analysis. The EcoRI restriction site was present in wtBQCV and not in mutBQCV.