A. Fulton; G.A. Perkins; S. Peters; N. Osterrieder; G.R. Van de Walle
RNA interference mediated by small interfering RNAs (siRNAs) is an important defense mechanism against viral infections in plants, and synthetic siRNAs have been shown to inhibit viral replication of human immunodeficiency virus, herpes simplex virus and others. siRNAs bind to complementary target mRNA resulting in degradation and inhibition of protein expression. Equine herpesvirus type 1 (EHV-1) causes myeloencephalitis, abortions and respiratory disease and spreads rapidly by aerosolization of the virus and inhalation. Vaccines against the disease are marginally efficacious and metaphylactic and therapeutic agents are not available. siRNAs, delivered nasally, could prove to be a novel and efficacious method of EHV-1 control. Effective siRNAs could be given 1) prior to co-mingling of horses at competitions, sales, and show events, and 2) during an EHV-1 outbreak to decrease viral load in the herd and prevent new infections. We investigated whether siRNAs against EHV-1 genes responsible for viral replication and cell entry would decrease EHV-1 infection, which was studied first in vitro and then in vivo.
In vitro: siRNAs against two essential EHV-1 genes were synthesized and transfected at various concentrations into rabbit kidney-13 cells, followed by infection with the neurovirulent EHV-1 strain Ab4. Cell culture supernatant was collected at 24-h post infection (p.i.) and the number of infectious units determined by plaque assays. In addition, the monolayers were examined to measure plaque sizes and morphology by fluorescent microscopy. Both siRNAs significantly reduced viral replication as measured by the magnitude of virus production and plaque size (p<0.05, Student's t-test), and a maximal effect was noted at 75nM with an 80-fold reduction. Interestingly, combining both siRNAs proved to be as effective as use of a single siRNA at 75 nM, but at a much lower concentration, namely 12.5 nM of each siRNA.
In vivo: Four-week old BALB/c mice were given various concentrations of the siRNAs, alone or in combination, intranasally and were subsequently infected with 105 PFU Ab4 at 0.5, 6, 12 and 24-h later. Treatment with siRNAs not directed against EHV-1 genes, the carrier (lipofectamine) and media were control groups. All mice were weighed daily for two weeks and the lungs were harvested (3 mice per group) on days 2 and 4 p.i. for viral titers. Mice treated with siRNAs 30-min and 6-h prior to EHV-1 infection lost less weight compared to the controls, and viral titers in the lungs were significantly lower (p<0.05, Student's t-test).
From these results, we conclude that siRNAs directed against two genes vital to EHV-1 replication are highly effective at down regulating viral replication in vitro and also in vivo in a mouse model of EHV-1 infection. Therefore, siRNAs show potential for preventing EHV-1 infection in horses. The efficacy of siRNAs in the definitive host, the horse, will be tested in the near future.