Document Type

Thesis - University Access Only

Award Date

2004

Degree Name

Master of Science (MS)

Department / School

Veterinary and Biomedical Sciences

First Advisor

Jane Christopher-Hennings

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is a widespread and economically devastating disease affecting the swine industry. The causative agent of PRRS is a single stranded positive sense RNA virus. Porcine reproductive and respiratory syndrome virus (PRRSV) is transmitted though insemination with infected semen, pig-to-pig contact, and aerosols.

In this study, a real-time PCR assay that targeted the 3'UTR region of the PRRSV genome, was developed for the detection and quantification of PRRSV in serum and semen. All samples were quantitated using a standard curve obtained by serial dilutions of an in-vitro transcript. The lower limit of sensitivity was consistently observed at approximately 33 copies/ml. No reactivity was noted with 9 non-related swine viruses. In addition, a modified RNA extraction was validated for use in semen to replace a phenol/chloroform method.

The next objective was to determine the quantity of PRRSV within semen and serum samples from experimentally infected boars, and determine whether there is a ·correlation between viral loads in serum and semen. In general, the concentration and duration of PRRSV shedding in semen did not correlate with quantity or duration in serum.

This study also determined the time course of shedding in boars that are persistently infected with PRRSV up to 96 days after infection. Viral RNA was not found in any serum and semen sample after 79 days post inoculation. However, PRRSV RNA was found in various lymphoid tissues in all of the principal boars. There was no detectable PRRSV RNA in any reproductive tissues.

The levels of gamma interferon in peripheral mononuclear cells were measured weekly through out the study, using an ELISPOT assay, in order to monitor the cellular immune response to PRRSV. Our results showed a peak gamma interferon response at 36 DPI that corresponded with the cessation of PRRSV detection in serum and semen samples in most boars. Gamma interferon levels in the peripheral circulation did not appear to prevent viral RNA persistence in lymphoid tissues.

The final objective was to determine whether PRRSV genetic changes might be related to viral persistence. The sequence within the PRRSV ORF 5 genome from the inoculation virus was compared to the RNA sequences recovered from serum and semen samples at 22 DPI and to the sequences from the tissues at 96 DPI. The nucleotide similarity between the inoculating virus sequence and the nucleotide sequence of the RNA recovered from the serum and semen ranged from 99.3 to 99.8%, while the nucleotide similarity to RNA recovered from tissues ranged from 98.8 to 99.8%. Due to high percent of nucleotide similarity, the persistence of the virus in this study does not appear to be due to substantial virus mutations.

In summary, we successfully developed a real-time PCR assay to detect and quantify PRRSV in both serum and semen samples. Lymphoid tissue was determined to be the site of PRRSV persistence in this study, as all boars had detectable PRRSV RNA in various lymphoid tissues at 96 DPI. Finally, the cessation of PRRSV in serum and semen may be related to gamma interferon production and appearance. In this study, the persistence of PRRSV did not appear to be from substantial viral genetic changes within the ORF 5 region, since the ORF 5 sequence obtained from the clinical specimens had a high percent nucleotide similarity to the sequence of the inoculating virus.

Library of Congress Subject Headings

Porcine reproductive and respiratory syndrome.
Boars -- Virus diseases.

Publisher

South Dakota State University

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Rights Statement

In Copyright