Date of Award
Doctor of Philosophy
Comparative and Experimental Medicine
Stephen A. Kania
Melissa A. Kennedy, Karla J. Matteson, Leon N.D. Potgieter
Feline herpesvirus 1 (FHV-1) is a linear double-stranded DNA virus that causes approximately 50% of the upper respiratory tract infections and produces the most severe respiratory disease in domestic cats. Primary ocular infection, as occurs in humans with the related virus, herpes simplex virus type 1 (HSV-1), consistently produces conjunctivitis and minimal corneal involvement; however, clinical manifestations of disease due to repeated recrudescence involve the cornea and can potentially lead to blindness. Vaccines only produce partial protection from clinical disease, and antiviral medications approved for treatment of HSV-1 in humans are only minimally effective for treatment of the chronic cases in cats. Therefore, RNA interference (RNAi), a RNA-guided gene regulatory mechanism that is found in a variety of eukaryotic organisms and provides anti-viral immunity in plants, was used as a therapeutic method for prevention of FHV-1 infection in feline cells. Previous RNAi experiments in related herpesviruses demonstrated that the DNA polymerase gene and a gene coding for a viral attachment protein are effective targets for inhibiting herpesvirus replication. Therefore, a region coding for highly conserved amino acid motifs of herpesvirus DNA polymerase genes, which is unique to each viral species and lacks DNA sequence drift for alphaherpesviruses, including herpes simplex virus type 2 and also feline herpesvirus as shown in this study, was targeted by RNAi. The attachment proteins for FHV-1 are unknown, but the highly conserved glycoprotein D (gD) gene was also chosen as a target for this study. HSV-1 gD is an essential receptor-binding polypeptide and is necessary for penetration of the virus into cells. FHV-1 gD, an envelope protein, is an inducer of virus-neutralizing antibodies and may play an important role in the restriction of the host range of the virus to feline cells.
Two synthetic siRNAs targeting the DNA polymerase gene and the gD gene of FHV-1 were effective in knockdown of their intended targets by 69-83% for DNA polymerase mRNA and 77-87% for gD mRNA, determined by quantitative real-time RT-PCR. Based on flow cytometry results, glycoprotein D mRNA knockdown decreased gD cell surface expression by 27-43%, and DNA polymerase mRNA interference decreased cell surface FHV-1 glycoprotein expression by 29-71%. Knockdown of the mRNAs from these genes also resulted in decreased infective virus in vitro, with decreased viral replication by 83-96% by DNA polymerase RNAi and 77-84% by gD RNAi, determined by plaque assays. Interference of each mRNA also resulted in a decrease in the amount of the opposite mRNA, independent of interferon β production. These results indicate that FHV-1 glycoprotein D, like its homolog in HSV-1, is essential for in vitro replication and is likely involved with viral attachment and/or penetration, and both this gene and the FHV-1 DNA polymerase gene are suitable targets for RNAi anti-viral treatment. This study lays the groundwork for potential in vivo investigations in cats. Such studies may provide unique insights into the prevention/treatment of herpesvirus infections by RNAi.
Wilkes, Rebecca Penrose, "RNA Interference of the Glycoprotein D and DNA Polymerase Genes of Feline Herpesvirus 1 by Synthetic siRNAs. " PhD diss., University of Tennessee, 2007.