Nucleotide sequencing of 5′ and 3′ termini of bovine viral diarrhea virus by RNA ligation and PCR

Abstract

Genomic RNA was extracted from cytopathic (CP) bovine viral diarrhea virus (BVDV) strain NADL, CP strain 72, and noncytopathic (NCP) strain SD-1 purified by ultracentrifugation. Assuming the presence of a cap structure, de-blocking of the 5′ capped end of the genomic RNA was done by treatment with tobacco acid pyrophosphatase (TAP). Following decapping, the RNA molecules were ligated using T4 RNA ligase and the ligated tandem RNA templates were then amplified by primer-directed amplification (PCR). cDNA synthesis was done using reverse transcriptase with random primers and cDNA amplification was done using a negative sense primer 231–248 and a positive sense primer 12434–12451. The nucleotide sequence of the amplified product was determined by double-stranded sequencing using the Sanger di-deoxy chain termination method and an additional ‘CCCCC’ nucleotide sequence was identified at the ligation site. Following dATP tailing of cDNA and amplification across the 5′ terminus and nucleotide sequencing, no additional nucleotides were identified on the 5′ terminus. The 5′ terminus as published by Collett et al., 1988b was confirmed as previously reported. Therefore, the 3′ terminus includes an additional ‘CCCCC’ nucleotide sequence to that previously reported. Identical results were obtained when the BVDV genomic RNA was not decapped prior to RNA ligation and amplification.