Document Type

Thesis - University Access Only

Award Date

2006

Degree Name

Doctor of Philosophy (PhD)

Department / School

Biology

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped, positive stranded RNA virus belonging to the Arteriviridae family and has emerged as the most economically important swine disease in the mid 1990' s. PRRSV exists as two major genotypes classified as either European-like (Type I) or North American (Type 2). Despite sharing morphological and structural similarities, the high degree of genetic and antigenic differences observed between the two genotypes indicate that PRRSV has evolved independently on two separate continents. Type 1 PRRSV has recently emerged in the United States. The genomic RNA sequence of the Type 1 PRRSV index strain EuroPRRSV, shares 95% nucleotide identity with the European prototype strain Lelystad while it shares only 60% with that of the North American type strain (VR-2332). The two most important features of PRRSV are that it replicates in pulmonary macrophages establishing an acute, symptomatic disease and that PRRSV evades the humoral immune response and establishes a persistent infection within lymphoid sites. The overall objective of our studies was to characterize the in vivo pathogenesis and virulence of both Type 1 and Type 2 PRRSV strains. In all three experimental chapters, experiments were designed to assess the clinical condition of animals as well as measure changes in viral load, antibody, and virus neutralization activity. Our first objective, outlined in the second chapter, was to perform a detailed pathological analysis of Type 1 isolates now circulating in the United States. More specifically, our aim was to evaluate neutralizing properties of sera from pigs experimentally infected with representative U.S. Type 1 isolates and to assess serological cross protection among different isolates. Our second objective, outlined in the third and forth chapters, was to examine the potential of PRRSV to persist in the host by developing a diagnostic, in situ hybridization tool to identify cell types within various tissues that support low-level viral replication. In addition, we were able to use the data to build a model of PRRSV replication throughout the various stages of disease. The following three studies describe the rationale, experimental design, results and future direction of the research. The goal of the first study was to evaluate the virus neutralizing and pathogenic properties of selected Type I isolates based upon their genetic differences, geographic isolation and clinical symptoms. We hypothesize that understanding differences in neutralization titers among PRRSV strains will lead to developing vaccines with a wide cross protection among other strains. Forty pigs aged 4-6 weeks were divided into 5 groups and inoculated with one of four different European-like isolates; the fifth group served as a negative control. Blood samples and tissues were collected and characterized for viral load, antibody response and persistence. Clinical signs and pathology were variable among groups and all animals seroconverted within two weeks. Neutralizing antibody response among homologous challenge isolates reached a titer of at least 1: 16 among all isolates and peaked upwards of 1: 128 by 56 dpi. This study was the first controlled study to report on the relative virulence, persistent potential and pathogenesis of diverse U.S. Type 1 PRRSV isolates. Next, since we believe that the cells supporting an acute PRRSV infection may be different from cells supporting a persistent PRRSV infection, we wanted to use in situ hybridization (ISH) to identify where the virus is localized in persistently infected pigs. The second study was designed to localize PRRSV replication in pigs during a long-term, persistent, congenitally contracted infection. At term, 34 piglets were farrowed from three sows infected at 95 days gestation with PRRSV isolate VR-2332. Blood and tissues were gathered and PRRSV replication was assessed using virus isolation (VI), polymerase chain reaction (PCR) and ISH. VI from lung tissue and serum was positive until 21 days post partum ( dpp ), and by day 60, pigs were negative for PRRSV in serum by VI. Between 60 and 132 dpp, pigs that were randomly sacrificed showed no sign of viral replication as detected by VI in lung and other non-lymphoid organs; however, in situ hybridization detected the presence of active viral replication in these tissues. Beyond 300 dpp, replication became undetectable in serum and tissues and did not correlate with the appearance of neutralizing antibody. In observing the course of PRRSV, we propose a model in which PRRSV can be divided into three phases of disease: first, an acute, multiorgan system infection; then, a transient phase where the virus is cleared from the lungs; and finally, a persistent phase where cells within lymphoid organs support PRRSV replication, which is sufficient to transmit the disease. The third and final study was to determine if our modified ISH method provided a more sensitive means of PRRSV detection in cells and tissues than immunohistochemistry. The major objective of this study was to identify sites of PRRSV replication in cells and tissues and to identify macrophages supporting PRRSV replication by co-localizing ISH positive cells with a human, macrophage-specific antibody used in immunohistochemistry. Our results showed that MARC-145 cells infected with PRRSV were detected after 6 hours post infection while immunohistochemical staining for the nucleocapsid protein was not detected until after 12 hours post infection. For the investigation of PR RSV replication in tissues, an open reading frame 7 (ORF-7) specific, cDNA probe was hybridized to formalin-fixed, paraffin-embedded tissue sections that were obtained from multiple tissues including lung, tonsil, spleen, thymus, lymph node, aorta, intestine, kidney nasal turbinate, liver and brain. Negative controls included RNase-treated tissue sections, tissues from mock infected pigs and hybridization using a heterologous, ORF-7 lactate dehydrogenase elevating virus (LDV) specific probe. We were able to show virus replication in all tissues, but most remarkably in the lung, kidney and lymphoid organs. These results also demonstrate the multi-organ system nature of PRRSV infection. Furthermore, the identification of macrophage cells supporting virus replication at 21 DPI suggests the presence of a persistent infection. The results of the three studies presented within this dissertation provide a fundamental pathogenic and diagnostic characterization of both PR RSV genotypes. The full length sequence analysis ofGP5 conducted during this study was a major step towards gaining a better insight into the evolution and antigenic diversity of PRRSV. As a result, many diagnostic laboratories have already modified their PCR assays to increase the specificity for the detection of European-like PRRSV. With other information gathered, future serological tests can be developed with the understanding there are other areas besides GP5 that that impart cross neutralizing protection. As a result, neutralizing monoclonal antibodies can be developed against other immunodorninant proteins expressed from different regions of the genome. The development and use of an ISH cDNA probe allowed us to build a model of PRRSV infectivity and follow viral tropism throughout a persistent infection. This experimental model allowed us to identify the emergence of virus subpopulations during persistence which possessed a mutation in the ectodomain of ORF5. These mutations may increase the tropism for replication in macrophage subpopulations that reside in lymphoid tissue. Finally, these studies have given us better understanding of PRRSV neutralizing activity and lymphoid tissue persistence, both of which are required for future development of effective vaccine and control strategies.

Library of Congress Subject Headings

Porcine reproductive and respiratory syndrome

Swine -- Virus diseases -- Pathogenesis

Format

application/pdf

Number of Pages

160

Publisher

South Dakota State University

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