Document Type

Thesis - University Access Only

Award Date

2002

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology

Abstract

Bovine viral diarrhea virus (BVDV), a small positive strand RNA virus, is a member of family Flaviviridae, genus pestivirus. BVDV is one of the most important pathogens of cattle, causing a variety of clinical syndromes, including abortion, digestive, respiratory and immunosuppressive diseases. Field isolates of BVDV can be divided into two biotypes cytopathic (CP) and noncytopathic (NCP) according to their ability to induce cytopathology in bovine cell cultures. Several hypotheses have been advanced to explain the cytopathology of BVDV; however the mechanism of BVDV cytopathogenicity remains unknown. The goal of this work was to investigate the mechanism involved in bovine viral diarrhea virus pathogenesis. Since phosphorylation of viral proteins, interferon (INF) response, and apoptosis are major factors in viral infection, we investigated these mechanisms to determine their role in BVDV cellular pathogenesis. Intra-cellular connections between these events are well established. First, we analyzed the role of phosphorylation in BVDV infection. Our results showed that the pattern and kinetics of phosphorylation did not differentiate between the biotypes. NS5A was phosphorylated in both biotypes. Quantitative analysis of NS5A phosphorylation and the effects of kinase inhibitors suggested a role of the phosphorylation in BVDV replication and cytopathogenicity. It was observed that: 1) NS5A phosphorylation levels were higher in strains of the CP than the NCP biotype; 2) Two protein kinase inhibitors (genistein and staurosporine) reduced viral replication (titers) of both biotypes, but had a greater effect on CP than NCP strains; and 3) Staurosporine inhibited NS5A phosphorylation in CP biotype more than the NCP strains. We investigated the effect of phosphorylation on nuclear localization of NS5A. Phosphorylated NS5A protein of both biotypes was identified only in the cytoplasm. Second, we analyzed the role of IFN in BVDV infection. Our result showed that IFNalpha greatly inhibited BVDV growth, up to 5 logs in CP and 2-3 logs in NCP strains. Using the most recent and powerful tool, RT-PCR, the antiviral effect of IFN-alpha was identified to be through inhibition of RNA synthesis. IFN-alpha inhibited RNA production by 4 logs in CP and 2 logs in NCP strains. The results also supported the hypothesis that RNA accumulation level was a factor in cytopathogenicity, where CP strains RNA level exceeded that of CP strains up to 2 logs. Finally, we studied the role of the p53 and caspase pathways in BVDV apoptosis. The results showed that pro-apoptotic factor, p53, is increased in CP strains infected cells after 48 h of infection; but the anti-apoptotic factor, Bcl2, was decreased in CP strains infected cells after 48 h of infection. There were no changes in the level of p53 or Bcl2 in NCP strains infected cells compared with non infected cells. Although caspase was not activated, caspase inhibitors decreased viral growth up to 1 log. The results indicate that p53 and caspase pathways are involved in BVDV apoptosis. The results obtained in this work increased our knowledge about the mechanism of BVDV pathogenesis. This knowledge will help in effective protective and control mechanism

Library of Congress Subject Headings

Bovine viral diarrhea -- Pathogenesis Cattle -- Virus diseases

Format

application/pdf

Number of Pages

177

Publisher

South Dakota State University

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