Doctoral Dissertations

Date of Award

5-2002

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

L. N. D. Potgieter

Committee Members

Stephen Kania, Melissa Kennedy, Karla Mattesson, Sarel Van Amstel

Abstract

Respiratory syncytial viruses (RSVs) in ruminants are classified into two subgroups, ovine RSV and bovine RSV. Although ovine RSV infects cattle, its contribution to bovine respiratory tract disease has not been established which is an important issue for vaccine development in cattle.

Diagnosis by virus isolation or serology has low or variable sensitivity and/or specificity and PCR has been recommended as a rapid and sensitive technique for RSV detection. Prior to this study, a laboratory test to differentiate between bovine and ovine RSVs did not exist.

First, the nucleotide sequence of the ovine RSV fusion (F) gene was determined and compared with representative strains of bovine RSV and human RSV subgroups A and B. The ovine RSV F gene has 85% and 72-73% nucleotide identity with those of bovine RSV and human RSV respectively. The predicted amino acid sequence of the ovine RSV F gene has 94% and 83-84% amino acid identity with those of bovine RSV and human RSV respectively.

Two RT-PCR assays have been developed for simultaneous detection of bovine and ovine respiratory syncytial viruses. One, a set of primers amplified a 426 bp fragment of either bovine or ovine RSV F gene (RT-PCR F). PCR-F products could be distinguished by EcoRI or BstYI restriction endonuclease cleavage. In the other assay, a set of primers amplified a 542 bp fragment of either ovine or bovine RSV G gene (RTPCR G). EcoO109I and RsaI restriction enzymes were used to differentiate between the ovine and bovine PCR-G products. Sequencing of the PCR products confirmed the fidelity of both assays.

The two assays were evaluated using eight bovine RSV isolates, one ovine RSV, one bighorn sheep RSV, one caprine RSV, two human RSV isolates and several viruses associated with bovine respiratory tract disease.

The two PCR assays described in this study potentially constitute sensitive and specific means for simultaneous detection of bovine and ovine RSV and differentiation between them. However, RT-PCR F followed by the appropriate restriction enzyme cleavage may be superior to RT-PCR G to discriminate between the two ruminant RSV subgroups and for determining the relative contribution of ovine and bovine RSV to the pathogenesis of bovine respiratory tract disease. This is relevant to the complete understanding of RSV epidemiology and immunoprophylaxis.

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