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.
Materials and Methods
Cell line and virus strains—Crandell-Rees feline kidney cellsa were propagated and maintained in culture medium,b as described elsewhere.28 A prototype virus, C-27 strainc of FHV-1, was used to infect the CRFK cells. A calicivirus wild-type strain was supplied by a university virology laboratoryd and used in the study.
siRNAs and transfection—Five siRNAse (DNA1 through DNA5, respectively; Appendix 1) were designed to target mRNA that code for the DNA polymerase gene of FHV-1. The regions of the mRNA of the FHV-1 DNA polymerase gene targeted by the siRNAs have sequences that are identical to each of the 8 DNA polymerase gene sequences available in the GenBank nucleotide sequence database and were identical in an additional 10 isolates from infected cats collected from 2003 to 2006 by the university virology laboratory.d These targeted sequences were compared with known sequences in the GenBank database to decrease off-target effects by avoiding similar sequences in the feline genome.
In 6-well plates,f CRFK cells were transfected with 100nM of siRNA/well and 5 μL/sample of a transfection reagent,g as described elsewhere.28 Control samples used in each experiment included mock-transfected, uninfected CRFK cells; nontransfected, FHV-1–infected CRFK cells; negative control siRNAh-transfected, FHV-1–infected CRFK cells; fluorescently labeled, siRNA-transfected controli CRFK cells; and nontransfected CRFK cells infected with feline calicivirusd (type 1 interferon control cells); the latter were included because interferon B is activated when CRFK cells are infected with an RNA virus (ie, feline calicivirus).28 Each siRNA was initially tested in duplicate, and functional siRNAs were tested 2 additional times for a total of 3 experiments/functional siRNA.
In 6-well plates,f experiments that included combinations of 2 siRNAs (G3 and gD1)28 that target the FHV-1 glycoprotein D gene mRNA and 2 siRNAs (DNA1 and DNA3) that target the FHV-1 DNA polymerase gene mRNA were performed with 6 μL of a polyamine mixturej/well in accordance with the manufacturer's recommendations. Combinations of 2 siRNAs/mixture were combined (50nM of each siRNA) for a total concentration of 100nM/well to transfect preplated CRFK cells with a confluency of approximately 80%. All possible combinations of the 4 siRNAs were tested. Each combination of siRNAs was evaluated in duplicate, and each experiment was repeated twice. Each of the 4 siRNAs was also tested individually in these experiments in 100nM total concentration/sample.
Virus infection and plaque assays—The C-27 strain of FHV-1 used for the study was grown in CRFK cells until a cytopathic effect in 50% of cells was detected via visual examination. Plaque-forming units were determined by use of a plaque assay, as described elsewhere,31 with modifications. Feline serum with an anti–FHV-1 antibody titer > 2,560 when tested by use of an immunofluorescent antibody assay was provided by the university virology laboratory.d The serum was diluted 1:50 and used as a substitute for the agarose overlay. Aliquots of virus were prepared and frozen at −80°C, and each aliquot was used only 1 time for each experiment.
Transfected CRFK cells were infected with FHV-1 at a multiplicity of infection (0.1), as described elsewhere.28 At 48 hours after infection with FHV-1, a 500-μL sample of cell culture medium was removed from each well and stored at −80°C until plaque assays could be performed.
Flow cytometry—At 48 hours after infection with FHV-1 or calicivirus, the CRFK cells in each test and control well were treated with 0.05% trypsin (1 mL), washed with 1 mL of PBS solution,k and resuspended with 1 mL of PBS solutionk/sample. One hundred microliters of the cell suspension was removed from each well and stored on ice until extraction of RNA was performed. An additional 100 μL of each suspension was removed and processed for western blot analysis. The remaining volume (800 μL) of each sample was processed for flow cytometric analysis.
Suspension samples of the CRFK cells collected for flow cytometric analysis were washed in a flow buffer (60 mL of 0.5% sodium azide solution, 87 mL of PBS solution, and 3 mL of fetal bovine serum), and the cells were pelleted by centrifugation at 1,000 × g. Two hundred microliters of a fluorescein isothiocyanate–labeled, cat anti–FHV-1 polyclonal antibodyl was applied to the CFRK cells of each sample and stored on ice for 1 hour. Each sample was washed in PBS solution and resuspended with 1 mL of PBS solution/sample. Cell surface fluorescence was assessed with a flow cytometer.m Transfection efficiency was also evaluated via flow cytometry by use of the fluorescently labeled siRNA control cells.i Cells were trypsinized and pelleted 24 hours following transfection, resuspended in 1 mL of PBS/sample, and evaluated with the flow cytometer.
Western blot analysis—The CRFK cell suspension samples were combined with SDS buffern and boiled for 5 minutes. Samples were stored at −20°C until assayed. Proteins were separated via PAGE by use of 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes.o The nitrocellulose membranes were probed with a polyclonal antibody.l Furthermore, an anti–glyceraldehyde 3-phosphate dehydrogenase monoclonal antibodyp was used as a loading control sample. Samples were incubated at 22° to 25°C for 1 hour with continual mixing by an orbital shaker. The nitrocellulose membranes were washed 5 times. Then, a 1:1,000 dilution of peroxidase-labeled, secondary antibodies (goat–anti-mouse IgGq [glyceraldehyde 3-phosphate dehydrogenase monoclonal] or goat–anti-feline IgGr [FHV polyclonal]) was applied to the washed membranes, and membranes were incubated at 22° to 25°C for 1 hour with continual mixing by an orbital shaker. Five additional washes were performed prior to detection of proteins by use of enhanced chemiluminescence.s
RNA extraction and real-time RT-PCR assay—The extraction of RNA and purification of RNA samples with a DNase treatment were performed, as described elsewhere.28 Purified RNA samples were stored at −80°C until analysis by use of the real-time RT-PCR assay. Primers and probes used for the RT-PCR assay were designed with a computer program32 for the detection of mRNA of the DNA polymerase gene of FHV-1 (Appendix 2). The primers and probe used for the detection of mRNA for interferon B have been described elsewhere.28 An internal control template (28S rRNA) was used for standardization of the RNA concentration per sample.34 Preparation of the reaction mixture for the RT-PCR assay was performed for each transcript by use of a kit,t as described elsewhere.28 The RT-PCR reaction for the detection of mRNA of the DNA polymerase gene of FHV-1 was conducted in a thermocycleru with the following temperature cycle conditions: an initial step at 45°C for 30 minutes to generate cDNA; a Taq polymerase activation step at 95°C for 2 minutes; and 45 cycles at 95°C for 15 seconds, 55°C for 60 seconds, and 72°C for 30 seconds. The temperature cycle conditions used for mRNA of interferon B and 28S rRNA have been described elsewhere.28 Formation of PCR product was monitored in real time by measuring the emitted fluorescence in the extension phase of the PCR cycles with the thermocycler system.u The software used by the thermocycler system qualified a reaction as positive for a target template when it detected 30 fluorescent units for the respective fluorescent emission channel. The PCR cycle at which this happened is regarded as the cycle threshold value, which is a template-concentration–dependent event. Three dilutions of standard FHV-1 DNA polymerase mRNA were included in each PCR run and used to generate standard curves for quantitation of initial template.
mRNA standards and standard curve construction—Messenger RNA standards were produced for FHV-1 DNA polymerase with techniques previously described.28 These standards were used to produce standard curves for absolute quantitation of FHV-1 DNA polymerase transcripts isolated from the samples. The RNA concentration and purity of the standards were determined by use of spectrophotometric analysisv (optical density, 260 and 280 nm). The number of mRNA copies in the stock solution was estimated on the basis of the molecular weight of the mRNA standards and the concentration of mRNA. Ten-fold serial dilutions of the mRNA stock solution were prepared, and aliquots of the dilutions were stored at −80°C. Each aliquot was used only once for analysis with the real-time RT-PCR assay.
The results from analysis with the real-time RT-PCR assay of 10-fold serial dilutions of the mRNA stock solutions were used to construct a standard curve. The intra-assay and interassay coefficients of variation for the reactions were determined from the results reported for the dilutions of the standard mRNA stock solution, as described elsewhere.35
The efficiency of the real-time RT-PCR assay to amplify the standard mRNA was evaluated with the following equation36:
where slope is a value derived from the constructed standard curves. Additionally, four 10-fold serial dilutions of RNA extracted from the nontransfected, FHV-1–infected control wells were prepared and tested in quadruplicate via the RT-PCR assay, as described previously, to calculate the amplification efficiency of the target RNA. This was done to ensure the standard RNA and the target RNA were amplified with similar efficiencies.
Cell viability assay—A cell proliferation assayw was used to determine any potential cellular toxicosis resulting from transfection of the CRFK cells. The CRFK cells were transfected individually with negative control siRNA, DNA1, and DNA3 with the cationic lipid transfection agent.g Additionally, cells were siRNA transfected individually with the combinations DNA1 + DNA3 and DNA1 + gD1 with the polyamine mixture.j At 24 hours after transfection, cells were assessed and compared with cells in nontransfected control wells.37 A product produced by metabolically active cells was detected by absorbance at 490 nm and was directly proportional to the number of live cells in the sample. Samples were tested in triplicate, and the experiment was performed twice.
Statistical analysis—Results for the 5 novel siRNAs and the control groups were compared via analysis with a univariate ANOVA method by use of a computer software package.x Furthermore, all assumptions were met for use of this statistical method, as determined by use of the Levene test of equality of variances and Shapiro-Wilk test of normality. Additionally, an ANOVA was used to compare siRNA combinations between treatment and control groups to evaluate logarithmic reduction in virus titer results. A least significant difference method was used for paired post hoc tests to compare between treatment groups, with α held to a simultaneous value of P < 0.05. The ANOVA and Bonferroni post hoc tests were used to evaluate siRNAs for cellular toxicosis. Values of P < 0.05 were considered significant.
Results
siRNA inhibtion of mRNA of the DNA polymerase gene of FHV-1—The RNAi directed against the mRNA of the DNA polymerase gene of FHV-1 was evaluated in CRFK cells. Cell transfection efficiency was evaluated with a fluorescently labeled siRNA control sample detected in CRFK cells by use of flow cytometry and was consistently 99% with both transfection protocols used (data not shown). Quantitative real-time RT-PCR was used to determine reduction of DNA polymerase mRNA resulting from siRNA treatment. The quantity of template mRNA copies of FHV-1 DNA polymerase gene was calculated for each sample from the linear standard curve (y = −3.406x + 40.38; R2 = 0.994); the curve represented concentrations of 6 orders of magnitude and resulted in a theoretical detection limit of 15 copies of template mRNA. The reproducibility of the real-time RT-PCR assay (intra-assay variation, 6% to 15%; interassay variation, 14% to 21%) was evaluated on the basis of the resulting cycle threshold values within the portion of the curve used to calculate the number of copies of mRNA that code for DNA polymerase. Amplification efficiency of the standard RNA was 97%, which was similar to the 96% amplification efficiency of the FHV-1 DNA polymerase mRNA. Only 2 (DNA1 and DNA3) of the 5 siRNAs targeting FHV-1 DNA polymerase mRNA were functional. Transfection of the FHV-1–infected CRFK cells with DNA1 and DNA3 significantly reduced the number of mRNA that code for DNA polymerase by mean ± SD values of 83 ± 6% and 69 ± 6%, respectively, compared with the number of mRNA produced in the control samples (Figure 1). The control value for these experiments was the mean of the RT-PCR assay results from the nontransfected, FHV-1–infected and negative siRNA-transfected, FHV-1–infected cells, which did not different significantly (P = 0.33) from each other. Furthemore, inhibition of mRNA for DNA polymerase by RNAi was independent of type 1 interferon production (Figure 2).
Comparison of the mean ± SD inhibition of mRNA of the FHV-1 DNA polymerase gene by the siRNAs DNA1 and DNA3. Values reported represent results from 3 experiments. The RNA was extracted from CRFK cells and analyzed by use of a real-time RT-PCR assay with primers and a probe specifically designed for detection of mRNA of the FHV-1 DNA polymerase gene. The concentration of RNA was normalized with that of 28S rRNA, and the number of template copies of mRNA of the DNA polymerase gene were estimated from a standard curve (y = −3.406x + 40.38; R2 = 0.994) generated via 10-fold serial dilutions of a DNA polymerase mRNA standard. There was a significant reduction in the copy number of mRNA for DNA1-treated (P = 0.006) and DNA3-treated (P = 0.015) cells, compared with results for the control sample (mean of the results from nontransfected, FHV-1–infected cells and negative control siRNA-transfected, FHV-1–infected cells).
Citation: American Journal of Veterinary Research 71, 6; 10.2460/ajvr.71.6.655
Interferon B real-time RT-PCR assay results. Reduction of FHV-1 DNA polymerase mRNA by RNAi was an siRNA-specific event and not the result of type 1 interferon induction. Interferon B mRNA copy numbers detected in cells infected with calicivirus (a), which is an interferon B inducer, are much higher than the copy numbers in samples treated with DNA1 or DNA3 (b), which were similar to the copy numbers detected in the mock-transfected CRFK control samples (b). Values reported represent results from a single representative experiment. Three separate experiments were performed, each of which had similar results.
Citation: American Journal of Veterinary Research 71, 6; 10.2460/ajvr.71.6.655
RNAi-induced reduction in FHV-1 protein production—Inhibition of mRNA used for DNA polymerase translation by DNA1 and DNA3 resulted in a decrease in production of FHV-1 proteins, compared with protein production for negative control siRNA-transfected, FHV-1–infected CFRK cells and nontransfected, FHV-1–infected CFRK cells. Total protein was isolated from the infected cells and analyzed by use of western blot techniques (Figure 3). Cells were also tested by use of flow cytometry to compare the quantity of FHV-1 glycoproteins on the surface of siRNA-transfected, FHV-1–infected CRFK cells with the quantity for negative control siRNA-transfected, FHV-1–infected CFRK cells (Figure 4). The quantity of surface glycoproteins was reduced by a mean ± SD value of 71 ± 4% in DNA1-transfected, FHV-1–infected CRFK cells and by 29 ± 8% in DNA3-transfected, FHV-1–infected CRFK cells, compared with the quantity of surface glycoproteins in negative control siRNA-transfected, FHV-1–infected CRFK cells. Combinations of siRNAs also reduced FHV-1 glycoproteins on the surface of infected cells. Transfection with 2 combinations of siRNAs (ie, DNA1 + DNA3 and DNA1 + gD1) significantly reduced the quantity of surface glycoproteins in FHV-1–infected CRFK cells by 72 ± 7% and 71 ± 6%, respectively (Figure 5). The DNA1 and DNA3 siRNAs were also tested independently in the combination experiments and caused a reduction in the FHV-1 surface glycoproteins equivalent to that detected in the initial experiments (data not shown).
Comparison of FHV-1 protein production by FHV-1 DNA polymerase mRNA-specific, siRNA-transfected, FHV-1–infected cells, and negative control siRNA-transfected, FHV-1–infected CRFK cells. The CRFK cells were transfected 24 hours prior to infection with FHV-1. At 48 hours after infection, cellular material was separated by PAGE on 10% SDS-polyacrylamide gels. Separated proteins were transferred to nitrocellulose membranes, and the membranes were probed with anti–FHV-1 polyclonal antibody (top gel) or an anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH) monoclonal antibody (bottom gel). Lanes were as follows: 1 = negative control siRNA-transfected, FHV-1–infected CRFK cells; 2 = mock-transfected, uninfected CRFK cells; 3 and 4 = DNA1-transfected, FHV-1–infected CRFK cells; and 5 and 6 = DNA3-transfected, FHV-1–infected CRFK cells.
Citation: American Journal of Veterinary Research 71, 6; 10.2460/ajvr.71.6.655
Flow cytometric evaluation of the production of FHV-1 proteins on the surface of negative control siRNA-transfected, FHV-1–infected CRFK cells (A); FHV-1–infected CRFK cells transfected with DNA1 (B) or DNA3 (C) 48 hours after FHV-1 infection (multiplicity of infection, 0.1); and mock-transfected, uninfected CRFK cells (D). Crandell-Rees feline kidney cells were incubated with polyclonal anti–FHV-1 antibodies, and 104 cells/sample were analyzed by use of flow cytometry. Data are representative of 3 experiments.
Citation: American Journal of Veterinary Research 71, 6; 10.2460/ajvr.71.6.655
Flow cytometric evaluation of siRNA combinations on the expression of genes of FHV-1 for the production of FHV-1 proteins on the surface of FHV-1–infected CRFK cells transfected with negative control siRNA cells (A), FHV-1–infected CRFK cells transfected with siRNA combinations of DNA1 + DNA3 (B) or DNA1 + gD1 (C) 24 hours prior to FHV-1 infection (multiplicity of infection, 0.1), and mock-transfected, uninfected CRFK cells (D). Forty-eight hours after FHV-1 infection, CRFK cells were incubated with polyclonal anti–FHV-1 antibodies, and 104cells/sample were analyzed by use of flow cytometry. Data are representative of 2 experiments.
Citation: American Journal of Veterinary Research 71, 6; 10.2460/ajvr.71.6.655
Inhibition of in vitro FHV-1 replication by siRNA-transfection—Infectious FHV-1 was detected in the cell culture supernatants of siRNA-treated cells and control cells by use of a plaque assay. Mean ± SD DNA1 and DNA3 inhibition of FHV-1 replication was 96 ± 3% and 83 ± 7%, respectively, compared with results for control cells (Figure 6).
Comparison of the mean ± SD virus titer in negative control siRNA-transfected and DNA polymerase mRNA-specific, siRNA-transfected CRFK cells infected with FHV-1. The CRFK cells were transfected with a negative control siRNA (control sample) or with a DNA polymerase-specific siRNA (DNA1 or DNA3) 24 hours prior to FHV-1 infection (multiplicity of infection, 0.1). Forty-eight hours after FHV-1 infection, cell culture supernatant was collected from each sample and tested in duplicate by use of a plaque assay. Data represent results for 3 experiments. The virus titers for the DNA1- and DNA3-treated samples are significantly (P < 0.001) different from the titers derived for the control sample. PFUs = Plaque-forming units.
Citation: American Journal of Veterinary Research 71, 6; 10.2460/ajvr.71.6.655
Effect of siRNA combinations on inhibition of in vitro FHV-1 replication—Several combinations of siRNAs were evaluated for reduction of FHV-1 replication (Figure 7). Each combination yielded results that differed significantly from those for the nontransfected, FHV-1–infected CRFK cells. Two combinations (DNA1 + gD1 and DNA1 + DNA3) caused a reduction in virus titer (96% and 97%, respectively), compared with results for nontransfected, FHV-1–infected CRFK cells. The DNA1 and DNA3 siRNAs were also tested independently in these experiments and yielded reductions in the percentage of viral replication equivalent to the values detected in the initial experiments.
Comparison of mean ± SD inhibition of viral replication in FHV-1–infected CRFK cells by paired combinations of siRNA that target mRNA of the FHV-1 DNA polymerase or glycoprotein D genes. Preplated cells were transfected with combinations of siRNAs targeting FHV-1 DNA polymerase (DNA1 and DNA3), glycoprotein D (G3 and gD1), or both 24 hours prior to infection of CRFK cells with FHV-1 (multiplicity of infection, 0.1). A 100nM concentration of siRNAs (50nM each) was used to transfect each well. Each combination was tested in duplicate. Forty-eight hours after FHV-1 infection, a sample of cell culture supernatant was collected from each well and tested by use of a plaque assay. Data represent results for 2 experiments. Inhibition of viral replication was significantly (P < 0.05) different from that of the nontransfected control sample (ie, FHV-1–infected CRFK cells; bar No. 1) for the siRNAs: DNA1 (bar No. 2), DNA1 + gD1 (bar No. 6), and DNA1 + DNA3 (bar No. 7) for causing the best inhibition of viral replication. PFUs = Plaque-forming units.
Citation: American Journal of Veterinary Research 71, 6; 10.2460/ajvr.71.6.655
Effect of transfection on cell viability—Transfection of cells with the negative control siRNA and DNA1 with the cationic lipid reagent had no detrimental effect on cell viability. The mean ± SD optical density of the control cells was 0.445 ± 0.017 and was not significantly (P = 1.0) different from the mean optical density of the cells transfected with negative control siRNA (0.450 ± 0.020) or from the cells transfected with DNA1 (0.454 ± 0.009). The DNA3 siRNA was determined (P < 0.001) to be slightly toxic when used at 100nM concentrations, with a 15% reduction in cell viability (mean ± SD optical density, 0.379 ± 0.018), compared with that in the control cells. However, when DNA3 was transfected with the polyamine mixture in combination with DNA1 (mean ± SD optical density, 0.428 ± 0.062), cell viability was not significantly (P = 0.066) different from that for the control cells (mean ± SD, 0.485 ± 0.015). The DNA1 transfected in combination with gD1 (mean ± SD optical density, 0.501 ± 0.022) was also not significantly (P = 1.0) different from that for the control cells.
Discussion
We used RNAi to target mRNA of the DNA polymerase gene of FHV-1. The use of RNAi caused a significant suppression of viral replication via inhibition of mRNA. On the basis of analysis of the study results, the FHV-1 DNA polymerase gene appears to be an excellent target for antiviral treatment by RNAi.
A localized respiratory tract infection is the disease manifestation in most cats with FHV-1.38 Therefore, the challenge of a targeted delivery through systemic administration of siRNAs could be eliminated. In vivo studies39 have revealed the effective uptake of siRNAs from mucosal surfaces, which includes effective intranasal delivery of siRNAs for the treatment of viral infections of the respiratory tract.40,41 In addition, RNAi is also effective in the treatment of ocular disease.24
A potential benefit of RNAi for the treatment of chronic FHV-1 ocular infections is the relatively long-term inhibition of mRNA. In the study reported here, inhibition continued at least 72 hours after transfection. Therefore, siRNAs could be used much less frequently than the current herpesvirus antiviral treatments that are applied 4 to 6 times/d.13
Inhibition can be enhanced by consecutive transfections with siRNAs into virus-infected cells. However, this can lead to selection of mutants with changes incorporated in the gene region that is being targeted,42 especially when treating chronic infections caused by RNA viruses populated by quasispecies populations of a virus.43 As a result of lower mutation frequencies of DNA viruses such as FHV-1, escape mutation is not expected to be as important a problem, compared with the problem of escape mutation in RNA viruses.44 Additionally, the targeting of conserved regions in essential genes is less likely to lead to mutational escape, and replication of the virus can be inhibited by targeting these genes.39
The targeting of conserved regions mitigates the problem of sequence diversity among viral isolates.39 Fortunately, FHV-1 apparently has a low rate of genetic variability among isolates.45 The region of the DNA polymerase gene targeted in the study reported here codes for highly conserved amino acid motifs of herpesvirus DNA polymerase genes, is unique to each viral species, and lacks DNA sequence drift for alphaherpesviruses, including herpes simplex virus type 246 and feline herpesvirus.
Development of escape mutants is reported42 to be prevented or decreased by use of a combination of siRNAs that target different parts of the viral genome. Additionally, inhibition of multiple genes is reported39 to be more effective than inhibition of a single gene. Therefore, we tested several combinations of siRNAs that target the DNA polymerase gene as well as the essential glycoprotein D gene.28 We discovered 2 combinations of siRNAs (DNA1 + DNA3 and DNA1 + gD1) that function as well as does DNA1, which was identified as the best single siRNA for suppressing viral replication and inhibiting glycoprotein production in FHV-1–infected CRFK cells. These combinations could potentially be used in the treatment of FHV-1 infections.
The study reported here provided important results that can be used for future in vivo investigations of FHV-1–induced disease in cats. In vivo studies may provide unique insights into the use of RNAi in the prevention and treatment of herpesvirus infection.
ABBREVIATIONS
| CRFK | Crandell-Rees feline kidney |
| FHV-1 | Feline herpesvirus-1 |
| RNAi | RNA interference |
| RT | Reverse transcriptase |
| siRNA | 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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Appendix 1
Design of the siRNAs used in experiments for targeting mRNA used for FHV-1 DNA polymerase gene translation.
| siRNA | Nucleotide sequenced*† | Location‡ |
|---|---|---|
| DNA1 | 5′-UGAGUAGCAUAUCUCUUCCta-3′ | 70–52 |
| DNA2 | 5′-GGUCAUAGCUUCUGGAAAAtc-3′ | 144–126 |
| DNA3 | 5′-UUUCGACUCCUUCUCUAGUtg-3′ | 121–103 |
| DNA4 | 5′-CUAGACUCCACGUAAUGUUta-3′ | 95–77 |
| DNA5 | 5′-AGACAUGGGAGAAGACCAUta-3′ | 23–5 |
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.
Appendix 2
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.
| Component | Nucleotide sequenced*† | Location‡ |
|---|---|---|
| Forward primer | 5′-CGGAGGGAAAATGCTTATGA-3′ | 3328–3347 |
| Reverse primer | 5′-ATCCATTCTCTGGGATGCAC-3′ | 3486–3467 |
| Probe | 5′-TCAATACATACGCTCGCCGATTAGTGGATA-3′ | 3387–3416 |
Nucleotide sequences are based on GenBank accession No. AJ224971.33
The primers and probe used were designed with a computer program32 for the detection of FHV-1 DNA polymerase.
See Appendix 1 for remainder of key.
