Feline herpesvirus-1 is a single, linear molecule of double-stranded DNA and a member of the subfamily Alphaherpesvirinae.1 There is widespread seroprevalence for FHV-1 in the feline population; as many as 71%2 to 97%3 of cats are seropositive for this virus. Furthermore, FHV-1 is reported3 as the etiologic agent for 50% of the cases of disease of the upper respiratory tract (ie, nasal cavity, nasopharynx, and trachea) and causes the most severe clinical upper respiratory tract disease in cats.4 Clinical signs include pyrexia, ocular and nasal discharge, rhinitis, tracheitis, and signs of depression.5,6 Conjunctivitis and minimal corneal involvement are clinical disease manifestations that are consistent with primary ocular infection caused by FHV-1; although potentially severe, the acute signs of disease typically resolve in a few weeks.4
Feline herpesvirus-1 has persisted as a result of the ability of this virus to establish lifelong latent infections.7,8 Approximately 80% of cats infected with FHV-1 become latently infected.7 During periods of stress (eg, housing changes, lactation, or parturition) or after corticosteroid administration, there is recrudescence of this disease, with an associated period of viral shedding that may not include clinical signs of disease. Furthermore, 29% of latently infected cats will spontaneously shed this virus.7 Although not all cats latently infected with FHV-1 will develop chronic clinical disease,9 a large percentage of adult cats do develop chronic disease.10 Important clinical manifestations of chronic clinical disease attributable to repeated FHV-1 recrudescence include corneal ulcers,11 eosinophilic keratitis, and corneal sequestration,12 and these manifestations can lead to blindness.9
Antiviral medications approved by the FDA for the treatment of infections caused by herpes simplex virus type 1 in humans are only minimally effective for the treatment of cats with chronic clinical disease caused by FHV-1.13 Of the acyclic nucleoside analogues available and that have been evaluated in cats, acyclovir is relatively ineffective against FHV-1 in vitro,14,15 and topically administered acyclovir has inadequate efficacy and requires application 5 times daily to be effective in the treatment of ocular disease caused by FHV-1.16 The in vitro use of ganciclovir and penciclovir has resulted in greater efficacy than has been detected for the use of acyclovir.15,17 Furthermore, treatment with famcyclovir, which is the oral prodrug form of penciclovir, is tolerated well by cats. However, pharmacokinetic profiles of famcyclovir in cats are complex; as a result, therapeutic drug concentrations of penciclovir in ocular tissues may not be achieved after oral administration.18
A potential concern regarding the use of nucleoside analogues is the development of thymidine kinase mutant viruses that are not susceptible to treatment with antiviral drugs. Furthermore, FHV-1 thymidine kinase mutant viruses resistant to penciclovir have been experimentally induced through the use of increasing concentrations of penciclovir.17 However, resistance to these drugs has been minimal in human patients with competent immune systems who were treated because of herpes simplex virus type 1 infections.19 An additional problem with currently available antiviral drugs is that these drugs are virustatic and not virucidal. Therefore, these drugs must be applied 4 to 6 times daily to be effective in the treatment of FHV-1. Idoxuridine, vidarabine, and trifluridine have been used in a clinical study13 as topical treatments for ocular lesions caused by herpes simplex virus type 1; however, the results of that study13 revealed that the response to these treatments is poor. A poor response to treatment may be the result of inadequate owner compliance for the frequency of application needed to enable these drugs to be effective.13
Cidofovir, a nucleoside monophosphate analogue of cytosine, has been effective against FHV-1 in in vitro studies.15,20 The extended half-life and persistence of drug in ocular tissues make cidofovir a good candidate as an antiviral treatment for herpesvirus-induced ocular disease in cats.21 Cidofovir was evaluated in an in vivo study,21 and although it was previously found to induce local adverse effects, such as ocular hypotonia and nasolacrimal duct obstruction in other species, these potential adverse effects were not investigated in cats.
Vaccines are available for immunization against FHV-1. As a result of the virus being poorly immunogenic,22 vaccines against FHV-1 do not prevent infection in seronegative cats or shedding of virus in infected cats; therefore, these vaccines only induce partial protection from clinical disease, and the development of a new treatment for FHV-1 would be beneficial.23
An RNAi mechanism has been manipulated for the prevention of various mammalian viral infections in vitro and in vivo.24 Ribonucleic acid interference is caused by a double-stranded, RNA-guided gene-silencing pathway that is found in a variety of eukaryotic organisms (ie, yeast, plants, and mammals).25 The double-stranded siRNAs that trigger this pathway can be supplied exogenously to inhibit expression of specific genes.25,26
Inhibition of viral replication is an optimal method for the inhibition of viral infection.27 Results of 1 study28 revealed that the targeting of an essential glycoprotein (ie, glycoprotein D) of FHV-1, which is transcribed late in infection, can effectively inhibit herpesvirus replication. However, targeting a gene, such as the gene for viral DNA polymerase, that is expressed earlier in the viral replication process is likely to result in an improvement in reduction of viral replication. Thus, targeting of essential genes of herpesviruses, including the FHV-1 DNA polymerase gene, can effectively inhibit herpesvirus replication.29 Five novel siRNAs have been designed to specifically target the DNA polymerase gene of FHV-1. The purpose of the study reported here was to evaluate the ability of these siRNAs to reduce in vitro viral replication and gene expression of FHV-1 by targeting the DNA polymerase gene, to evaluate combinations of siRNAs that target mRNA of the glycoprotein D or FHV-1 DNA polymerase genes, and to determine the combination or combinations of siRNAs that yield the greatest inhibition of in vitro FHV-1 replication.
Crandell-Rees feline kidney
Small interfering RNA
Crandell-Rees feline kidney cell line, American Type Culture Collection, Manassas, Va.
Dulbecco's minimal essential medium, Cambrex, Charles City, Iowa.
FHV-1 strain C-27, American Type Culture Collection, Manassas, Va.
Clinical Virology Laboratory, Department of Comparative Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville, Tenn.
Custom designed siRNAs, Ambion-Applied Biosystems, Austin, Tex.
Six-well cell culture cluster tissue culture treated plates, Corning Inc, Corning, NY.
Lipofectamine 2000, Invitrogen, Carlsbad, Calif.
Silencer negative control No. 1 siRNA, Applied Biosystems, Carlsbad, Calif.
Cy 3 labeled negative control No. 1 siRNA, Applied Biosystems, Carlsbad, Calif.
siPORT amine, Ambion-Applied Biosystems, Austin, Tex.
Invitrogen, Carlsbad, Calif.
Feline Herpesvirus FITC FA conjugate, Accurate Chemical and Scientific, Westbury, NY.
Epics XL, Beckman Coulter, Fullerton, Calif.
Sigma-Aldrich, St Louis, Mo.
Trans-Blot Transfer Medium, Bio-Rad, Hercules, Calif.
Anti-GAPDH, mouse monoclonal antibody, Ambion-Applied Biosystems, Austin, Tex.
Goat anti-mouse IgG (gamma), FITC labeled antibody, KPL, Gaithersburg, Md.
Anti-cat IgG (gamma), FITC labeled antibody, KPL, Gaithersburg, Md.
ECL western blotting analysis system, Amersham Biosciences, Piscataway, NJ.
SuperScript III Platinum One-Step qRT-PCR kit, Invitrogen, Carlsbad, Calif.
SmartCycler II, Cepheid, Sunnyvale, Calif.
BioPhotometer 6131, Eppendorf, Westbury, NY.
CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega, Madison, Wis.
SPSS Software, SPSS Inc, Chicago, Ill.
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Design of the siRNAs used in experiments for targeting mRNA used for FHV-1 DNA polymerase gene translation.
Nucleotide sequences are based on GenBank accession No. AF079125.30
Uppercase letters represent the guide nucleotide sequence, and lowercase letters represent the 3’ overhang.
Represents base pair range of the nucleotide sequence.
Design of the primers and probe used in the real-time RT-PCR assay for the detection of mRNA of the DNA polymerase gene of FHV-1.