Date of Award

12-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Melissa Kennedy

Committee Members

Stephen A. Kania, Robert Donnell, Karla J.Matteson, Rebecca P. Wilkes

Abstract

Feline corononavirus (FCoV) infection is ubiquitous in domestic cat populations worldwide and is usually associated with subclinical or mild enteritis. However, in some cats infection may result in the development of a fatal progressive disease called feline infectious peritonitis (FIP). FIP is considered to be the major cause of infectious-related death in pet cats. Currently, there is no protective vaccine or curative treatment to this highly fatal disease. In this study, we evaluated the ability of small interfering RNAs (siRNAs) to inhibit the in vitro viral replication and gene expression of FCoV as a potential treatment for FIP.

Five synthetic siRNAs were designed to target different regions of the FCoV genome. The siRNAs were tested individually and in various combinations in vitro for their antiviral effects against 2 strains of FCoV (feline infectious peritonitis virus WSU 79-1146 and feline enteric coronavirus WSU 79-1683). Tested combinations targeted the FCoV leader and 3′ untranslated region; FCoV leader region and nucleocapsid gene; and FCoV leader, 3′ untranslated region, and nucleocapsid gene. For each test condition, assessments included relative quantification of the inhibition of intracellular viral genomic RNA synthesis by means of real-time, reverse-transcription Polymerase chain reaction (PCR) analysis; flow cytometric evaluation of the reduction of viral protein expression in infected cells; and assessment of virus replication inhibition via titration of extracellular virus with a 50% tissue culture infective dose (TCID50) assay.

The 5 siRNAs had variable inhibitory effects on FCoV when used singly. Combinations of siRNAs that targeted different regions of the viral genome resulted in more effective viral inhibition than did individual siRNAs that targeted a single gene. The tested siRNA combinations resulted in approximately 95% reduction in viral replication (based on virus titration results), compared with findings in negative control non-targeting siRNA-treated FCoV-infected cells. This study shows that FCoV replication can be specifically inhibited by siRNAs that target coding and noncoding regions of the viral genome, suggesting a potential therapeutic application of RNA interference in treatment of feline infectious peritonitis.

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