Taeg Su Kim

Document Type

Thesis - University Access Only

Award Date

2001

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is a viral disease of swine, resulting in severe reproductive failure in sows and respiratory disease in young pigs. PRRSV is known as inducing apoptosis in uninfected cells during infection in vivo and in vitro via an unknown mechanism. Little is known about the fate of cells that have detached during PRRSV infection in cultures. Therefore, the cell death mechanism, either necrosis or apoptosis, during PRRSV infection of MARC-145 cells was studied in both adherent cells and detached cells in cultures. We did not observe the changes indicative of apoptosis among the adherent MARC-145 cells. However, ample evidence of apoptosis was present among the detached cells. Evidence of apoptotic cell death in detached cells included chromatin condensation, intemucleosomal DNA cleavage assayed by agarose gel electrophoresis and TUNEL. In addition, over 90 % of detached cells were infected with PRRSV, and positive for trypan blue. From this experiment, we suggest that apoptosis induced during PRRSV infection occurs in infected cells by a direct mechanism, however, the result is a necrotic cell death. The production of apoptotic cytokines during PRRSV infection of pigs was studied using a reverse-transcription polymerase chain reaction (RT-PCR) method. Oligonucleotide primers were designed to amplify porcine cytokine transcripts including IL-la, IL-2, Il-4, IL-6, IL-8, IL-10, IL-12, TNF-a, TNF-j3, IFN-y and the housekeeping gene 132-microglobulin. To validate RT-PCR, total mRNA extracted from unstimulated and mitogen-stimulated porcine peripheral blood mononuclear cells (PBMCs) was reverse-transcribed and amplified. Specificity of PCR-products was confirmed by sequencing. Band intensity of PCR products quantified by densitometric scanning was normalized against j32-microglobulin. Mitogen-stimulated lymphocytes showed elevating in IL-2, IL-4, IL-6 and IFN-y transcripts. PBMCs stimulated with LPS increased expression of inflammatory cytokines, IL-la and TNF-a. RT-PCR was also used to study cytokine gene expression in tissues. Pigs were infected with PRRSV, and total mRNA from lymph nodes and lungs was isolated and amplified. These results showed elevations in IFN-y, and TNF-a, two cytokines associated with apoptosis in inflammatory regions.

Library of Congress Subject Headings

Swine -- Virus diseases
Apoptosis Porcine reproductive and respiratory syndrome

Format

application/pdf

Number of Pages

83

Publisher

South Dakota State University

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