Molecular cloning of complementary DNA from a pneumopathic strain of bovine viral diarrhea virus and its diagnostic application.

摘要

A pneumopathic strain of bovine viral diarrhea virus was grown in cell culture and purified. Genomic ribonucleic acid was extracted, polyadenylated at the 3' end, and copied into complementary DNA after oligo-dT priming. Complementary DNA was male double stranded and cloned into the pUC9 plasmid. Approximately 200 complementary DNA clones varying in length from 0.5 to 2.5 kilobases were obtained. Hybridization assays indicated that the sequences isolated were specific for bovine viral diarrhea virus and that at least 5.5 kilobases of bovine viral diarrhea virus genome was represented in the library of complementary DNA clones, the majority of which may have originated from the 3' end of the virus genome. One cloned complementary DNA sequence was used as a 32P-labelled hybridization probe for bovine viral diarrhea virus detection. The probe hybridized with all cytopathic and noncytopathic strains of bovine viral diarrhea virus tested and was 100 times more sensitive than infectivity assays for the detection of bovine viral diarrhea virus. Hybridization did not occur with nucleic acids from bovine coronavirus, bluetongue virus, bovine adenovirus or uninfected cell cultures. Native plasmid DNA sequences, labelled with 32P, did not hybridize with bovine viral diarrhea virus ribonucleic acid.