Document Type

Thesis - University Access Only

Award Date

1997

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

Abstract

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is recognized as a devastating viral disease of swine throughout the world, causing tremendous economic loss. PRRSV is a small, enveloped, single-stranded positive sense RNA virus with 8 open reading frames (ORFs) which encode at least three major structural proteins: a nucleocapsid protein (N) (ORF 7), a nonglycosylated membrane protein (M) (ORF 6) and an envelope glycoprotein (E) (ORF5). Replication of PRRSV in MARC-145 cells can be evaluated ultrastructurally using immuno-gold electron microscopy, leading to greater insight into PRRSV morphogenesis. A modified method for culturing MARC-145 cells in BEEM capsules was used to grow monolayers of MARC-145 cells. These monolayers were then inoculated with either a us or European PRRSV isolate and processed for immuno-gold labeling (IEM) at 1, 2, 4, 8, 16, 24, 48, 72 hours and 7 days post-inoculation (PI). PRRSV infected MARC-145 cells were labeled after processing for transmission electron microscopy with antibodies conjugated with gold. Each antibody was specific for one of the three major virion structural proteins. Using the gold conjugated antibodies allowed direct visualization of intracellular location of these structural proteins during viral replication. IEM for the N protein indicated nucleocapsids were present within the nucleus and perinuclear regions from 1 hour to 72 hours PI; the M protein was localized in vesicles of the endoplasmic reticulum at 8 to 72 hours PI; and the E protein was observed 8 to 72 hours PI in the golgi complex. By 7 days PI most of the MARC-145 cells had lysed and insufficient numbers of cells were available for analysis. This new, modified method of culturing, inoculating and embedding cells in BEEM capsules for IEM was extremely valuable for ultrastructure preservation. Collectively, these results indicate that the N protein is produced early in infection followed by the Mand E proteins. Cellular localization of each protein indicates that PRRS virions are assembled in the cytoplasm, mature in the golgi apparatus and are released by exocytosis.

Library of Congress Subject Headings

Swine -- Virus diseases

Morphogenesis

Immunogold labeling

Electron microscopy

Porcine reproductive and respiratory syndrome

Format

application/pdf

Number of Pages

61

Publisher

South Dakota State University

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