Bovine viral diarrhea virus : molecular cloning of genomic RNA and its diagnostic application

Author

Kenny Virgil Brock

Date of Award

12-1987

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

L. N. D. Potgieter

Committee Members

David A. Brian, Barry T. Rouse, Roger Carrol

Abstract

Molecular cloning of a field isolate of bovine viral diarrhea virus (BVDV) strain 72 RNA was done in this study. The sensitivity and specificity of cloned cDNA sequences in hybridization assays with various BVDV strains were determined.

BVDV was purified from infected cell culture supernates by isopycnic density-gradient centrifugation. The density of the virions was 1.138 gm/cm³. Approximately 100 ug of BVDV genomic RNA was extracted from virus purified from 500 ml of infected cell culture fluids. BVDV genomic RNA then was tailed with adenine residues at the 3' end using poly-A polymerase. cDNA was synthesized from polyadenylated BVDV RNA templates with oligo-dT primers, reverse transcriptase, and DNA polymerase I. The newly synthesized double-stranded BVDV cDNA was C-tailed with terminal deoxytransferase and annealed into G-tailed, Pst-l-cut pUC9 plasmid. Escherichia coli was transformed with the recombinant plasmids and a library of approximately 200 BVDV specific cDNA clones varying in length from 0.5 to 2.6 kilobases were isolated.

The sensitivity and specificity of hybridization between the labelled cDNA and BVDV target sequences were determined. Cloned BVDV sequences were isolated from pUC9 plasmid DNA and labelled with 32_p by nick translation. The detection limit by dot blot hybridization assay was 20 pg of purified genomic BVDV RNA. cDNA hybridization probes were specific for all strains of BVDV tested, regardless of whether they were noncytopathic and cytopathic, but did not hybridize with heterologous bovine viruses tested. Probes did not hybridize with uninfected cell culture or cellular RNA. Hybridization probes were at least as sensitive as infectivity assays in detecting homologous virus.

Recommended Citation

Brock, Kenny Virgil, "Bovine viral diarrhea virus : molecular cloning of genomic RNA and its diagnostic application. " PhD diss., University of Tennessee, 1987.
https://trace.tennessee.edu/utk_graddiss/12024