Degree Type

Dissertation

Date of Award

2001

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

First Advisor

Jeffrey J. Zimmerman

Second Advisor

Kyoung-Jin Yoon

Abstract

Porcine reproductive and respiratory syndrome (PRRS) viruses are recognized as possessing a high degree of genetic and antigenic variability. Virus diversity has led to questions regarding the association of virus mutation and persistent infection in the host and raised concerns vis-a-vis protective immunity, the ability of diagnostic assays to detect novel variants, and the emergence of virulent strains. The purpose of this study was to assess on-going changes in PRRS virus during replication in pigs under experimental conditions.;Animals were inoculated with a plaque-cloned virus derived from VR-2332, the North American PRRS virus prototype, and 3 independent lines of in vivo replication were maintained for 367 days by pig-to-pig passage of virus at 60 day intervals. A total of 315 plaque-cloned viruses were recovered from pigs over the 367 day observation period and compared to the original plaque-cloned virus by sequencing of open reading frames (ORFs) 1b (replicase), 5 (major envelope protein), and 7 (nucleocapsid) of the genome. In addition, a one-way virus neutralization assay was performed to detect escape mutant variants and a panel of monoclonal antibodies, including five monoclonal antibodies against the nucleocapsid protein and two against the envelope protein, were used to screen for viral epitopic changes.;Virus variants were detectable by day 7 post inoculation and multiple variants were present concurrently in every pig sampled over the observation period. Over the course of 367 days of virus evolution, the mean mutation rate in ORFs 1 b, 5, and 7 was estimated at 6.7, 7.0, and 1.3 changes per 1000 nucleotides, or 0.2, 12.4, and 1.1 per 1000 amino acids. Thus, sequence analysis showed ORFs 1 b and 7 to be highly conserved at the amino acid level relative to ORF 5. Among the 315 virus clones, a total of 48 nucleotide and 22 amino acid ORF 5 variants were detected, with most of the mutations occurring as substitutions. Although the virus neutralization assay and monoclonal antibody analysis detected no escape mutant viruses or epitopic changes, PRRS virus was shown to evolve continuously during replication in infected pigs, with different genes of the viral genome facing differing degrees of positive and/or negative selection.

DOI

https://doi.org/10.31274/rtd-180813-11126

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu

Copyright Owner

Chih-Cheng Chang

Language

en

Proquest ID

AAI3016695

File Format

application/pdf

File Size

97 pages

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