Generation of full-length cDNA of the two genomic dsRNA segments of infectious bursal disease virus

H.J. Boot, A.H.M. ter Huurne, B.P.H. Peeters

    Research output: Contribution to journalArticleAcademicpeer-review

    23 Citations (Scopus)

    Abstract

    To determine the complete nucleotide sequence of Infectious Bursal Disease virus (IBDV) isolates, an efficient method was developed to generate full-length cDNA of both the genomic A- and B-segments. Reverse transcription was carried out at the highest possible temperature (50°C) for the reverse transcriptase enzyme, and the single stranded cDNA was subsequently amplified by using an optimized PCR. The double stranded, full-length cDNA was efficiently cloned into a high copy number plasmid. Our results show that the entire cDNA of both the A- and B-segment of a classical attenuated isolate (CEF94), and a very virulent field isolate (D6948), can be cloned. The method will simplify greatly the procedure to generate full-length cDNA and determine the nucleotide sequence of the entire genome of IBDV isolates.
    Original languageEnglish
    Pages (from-to)49-58
    JournalJournal of Virological Methods
    Volume84
    Issue number1
    DOIs
    Publication statusPublished - 2000

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