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Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut
West Nile virus causes fatal encephalitis in humans, mice, and other vertebrates. In the present study, we demonstrate that small interfering RNAs (siRNAs) inhibit West Nile virus replication in vitro. Moreover, the administration of siRNAs to mice by hydrodynamic injection 24 h before challenge with an intraperitoneal inoculum of West Nile virus reduced the viral load and afforded partial protection from lethal infection. These data show the efficacy of the prophylactic use of siRNAs against a viral infection in vivo and suggest new strategies to combat West Nile virus.
West Nile virus, a single-stranded RNA virus in the family Flaviviridae, was initially described in Africa and has recently appeared in North America [1, 2]. West Nile virus is maintained in an enzootic cycle that includes birds and mosquitoes. Since the first outbreak in the New York City area in 1999, West Nile virus has spread throughout much of North America, causing significant morbidity and mortality [3]. Effective drugs or vaccines to prevent West Nile virus infection have not yet been approved for use in humans. Experimentally infected mice typically die of infection with West Nile virus, thereby partially mimicking human disease and providing a model system in which to study viral pathogenesis and immunity.
RNA interference (RNAi) is a process of sequence-specific degradation of RNA in the cytoplasm of eukaryotic cells that is induced by double-stranded RNA. This phenomenon was first described in the nematode Caenorhabditis elegans [4] and is conserved in mammalian cells. RNAi is believed to act as a natural defense against incoming viruses and the expression of transposable elements, and it may potentially be used to treat a variety of diseases [4, 5]. Small interfering RNAs (siRNAs) have suppressed the expression of specific genes both in vitro and in experimental animal models [610]. siRNAs are double-stranded RNAs that associate with a multiprotein RNA-induced silencing complex (RISC), which targets sequence-specific degradation of complementary messenger RNAs, leading to the posttranscriptional inhibition of protein synthesis [11, 12]. Virus-specific siRNAs have been shown to inhibit viral replication in cell culture models of HIV [13, 14, 15], hepatitis B virus (HBV) and hepatitis C virus (HCV) [1618], influenza viruses [19], -herpesvirus [20], human papillomavirus [21], dengue viruses [22], and West Nile virus [23]. RNAi has also been used to target sequences from HCV [10] and inhibit the production of the Fas receptor in mice [8]. McCaffrey et al. demonstrated that short hairpin RNAs blocked replication of an HBV plasmid in mice and thereby identified a model for viral infection [9]. Recently, 2 research groups demonstrated that siRNAs or DNA vectors produced short hairpin RNAs that could reduce the influenza viral load in murine lungs and increase survival [24, 25]. These findings suggest that RNAi may be used to combat infectious agents.
MATERIALS AND METHODS
Viral isolates, cell lines, and mice.
West Nile viral isolate 2471 was provided by John F. Anderson at the Connecticut Agricultural Experiment Station. Vero cells (African green monkey kidney cell line ATCC CCL-81) were maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 1% L-glutamine, 1% penicillin-streptomycin, and 10% fetal bovine serum. Ten-week-old female BALB/c mice were purchased from the Jackson Laboratory.
siRNA transfection.
siRNA transfections were performed according to the instructions in the Silencer siRNA Transfection Kit Users' Manual (Ambion). The optimized conditions were 2 × 105 cells, 8 L of siPORT Amine siRNA Transfection Reagent (Ambion), and 5 L of 20 mmol/L siRNA in each well of a 6-well plate.
Viral infection.
Twenty-four hours after siRNA transfection, Vero cells were infected with West Nile virus. Two hundred thousand plaque-forming units of viral isolate (MOI 1) were added to the Vero cell medium for 24 h. Mice were challenged with 80 pfu of viral isolate in 100 L of PBS by intraperitoneal injection.
Total RNA extraction and first-strand cDNA reverse transcription.
Total RNA was extracted from Vero cells and murine tissues by use of the RNeasy Mini Kit (Qiagen). Murine tissues were completely homogenized in 600 L of RLT buffer by use of Mini-BeadBeater (Biospec Products). Any residual DNA was removed by use of RNase-free DNase. First-strand cDNA was synthesized by use of random primers and the ProSTAR first-strand reverse-transcription polymerase chain reaction (PCR) kit (Stratagene).
Quantitative PCR (Q-PCR) for the West Nile virus e and -actin genes.
The level of West Nile virus e gene RNA was compared with that of a normalized level of -actin RNA in the Q-PCR analysis. Specific primers for the e gene (5-TTC TCG AAG GCG ACA GCT G-3 and 5-CCG CCT CCA TAT TCA TCA TC-3 ) and a probe for the e gene (5-6FAM-ATG TCT AAG GAC CCT ACC ATC-TAMRA-3) were designed. Specific primers for the -actin gene in Vero cells (5-AGC GGG AAA TCG TGC GTG AC-3 and 5-CAA TGG TGA TGA CCT GGC CA-3 ) and a probe for the -actin gene in Vero cells (5-6FAM-CAC GGC GGC TTC TAG CTC CTC CC-TAMRA-3) were designed. Specific primers for the -actin gene in mice (5-AGA GGG AAA TCG TGC GTG AC-3 and 5-CAA TAG TGA TGA TGA CCT GGC CGT-3 ) and a probe for the -actin gene in mice (5-6FAM-CAC TGC CGC ATC CTC TTC CTC CC-TAMRA-3) were designed.
Hydrodynamic injection.
A hydrodynamic-based transfection method was used to inject 1.6 mL of Ringer's solution (147 mmol/L NaCl, 4 mmol/L KCl, and 1.13 mmol/L CaCl2) containing 180 g of siRNA into the tail veins of BALB/c mice within 5 s [31, 32].
Confocal immunofluorescence analysis.
Mice were transfected with Cy5-labeled siRNA, and samples of tissue were stained with propidium iodide and were analyzed at 48 h after hydrodynamic injection.
Plaque-forming assay.
Samples of tissue (50 mg) were completely homogenized in 500 L of PBS by use of Mini-BeadBeater. After low speed centrifugation, 200 L of the supernatant was added to 95%100% confluent Vero cells in 6-well plates and was incubated at 37°C and 5% CO2 for 1 h. The plates were washed with PBS to remove the tissue-containing buffer, were overlaid with 3 mL of DMEM-agarose, were incubated for 4 days, and were stained with 4% (vol/vol) Neutral Red. The plaques were then counted.
Statistical analyses.
Comparisons of survival curves were done by use of Kaplan-Meier estimates (log-rank test). Viral loads in Vero cells were compared by use of 1-way analysis of variance and then the Tukey test. Results for viral loads or titers in murine tissues were compared by use of a 2-tailed Fisher's exact test or Mann-Whitney test. Statistical analyses were done by use of GraphPad Prism (version 4.0; GraphPad software).
RESULTS
To determine whether siRNAs could inhibit West Nile virus replication in vitro, we first examined the transfection efficiency of Cy5-labeled siRNA in Vero cells. At 24 h, 90% ± 1% (mean ± SD) of the cells were transfected by Cy5-labeled siRNA (figure 2A and 2B). Next, experiments were performed in which Vero cells were transfected with siRNAs specific for the West Nile virus e gene, and 24 h later West Nile virus (2 × 105 pfu) was added to the medium. Cells were collected at 24 h, total RNA was extracted and reverse transcribed into cDNA, and the viral load was determined by use of Q-PCR. The results show that siRNAs W86 and W246 (figure 2C) inhibited West Nile virus replication by 76% ± 14% (P < .05) and 62% ± 12% (P > .05 [not significant]), respectively, compared with inhibition by control siRNA.
We then tested whether siRNAs W86 and W246 could alter the course of West Nile virus infection in vivo. To monitor the distribution of injected siRNA, 6 mice were injected with Cy5-labeled siRNA. At day 2, these Cy5-labeled siRNA-transfected control mice were killed, and the livers and spleens were harvested. Confocal microscopy showed that siRNA could be detected in these tissues (figure 3A3D) and that transfection was therefore successful. Experiments with siRNAs specific for West Nile virus were then performed. At 24 h after injection with either siRNA W86, siRNA W246, or control siRNA, mice were challenged with 80 pfu of West Nile virus. Laboratory mice infected with West Nile virus typically develop a disseminated infection after several days and usually die at 712 days [29]. The siRNA W86, siRNA W246, and control siRNA groups had 31, 14, and 30 mice, respectively. Sixty-eight percent of the siRNA W86treated mice survived (P < .005), compared with 37% of the control siRNAtreated mice and 21% of the siRNA W246treated mice (figure 4). All the surviving mice appeared healthy at 4 weeks after viral challenge. These data suggest that siRNA W86 can alter the course of West Nile virus infection in mice and provide partial protection against fatal disease.
We then determined whether the viral load in the siRNA W86treated mice was lower than that in the control siRNAtreated mice. At days 3 and 6 after viral challenge, 9 experimental mice and 9 control mice were killed, and the livers, spleens, and brains from each mouse were examined for the presence of West Nile virus by use of Q-PCR and, in some cases, by use of a plaque-forming assay. Q-PCR was the primary method used to assess viral load, because Q-PCR directly correlates with traditional plaque-forming assays for the assessment of West Nile viral load, and because it is more sensitive than traditional plaque-forming assays [33, 34]. At day 3, West Nile virus was not detected by Q-PCR in the livers of the 9 siRNA W86treated mice, but it was detected in 5 of 9 control siRNAtreated mice (P < .05) (figure 5A). At day 6, virus was not evident in the livers of either group. At days 3 and 6, the viral load was lower in the spleens of the siRNA W86treated mice than in the spleens of the control siRNAtreated mice but the differences were not statistically significant (figure 5B and 5C). At day 6, West Nile virus could be detected, by Q-PCR, in the brains of 5 of 9 control siRNAtreated mice (Q-PCR mean value, 11 copies of e gene/1000 copies of -actin gene) and in the brains of 3 of 9 siRNA W86treated mice (Q-PCR mean value, 6 copies of e gene/1000 copies of -actin gene) (figure 5D). Plaque-forming assays were performed on 6 brain samples, and virus could be detected in 3 control siRNAtreated mice and in 3 siRNA W86treated mice. At day 6, the viral load was higher in the brains of control siRNAtreated mice (mean value, 131 pfu/g) than in the brains of siRNA W86treated mice (mean value, 29 pfu/g), but the difference was not statistically significant (figure 5E), which is a similar result to that of the Q-PCR assay. The viral loads in the livers, spleens, and brains of siRNA W246treated mice were similar to those in the control siRNAtreated mice (data not shown).
Studies were then undertaken to determine whether siRNA W86 could influence infection dynamics when administered after viral challenge. siRNA W86 or control siRNA was injected into mice 24 h after the administration of West Nile virus. siRNA W86treated mice (13 animals) and control siRNAtreated mice (15 animals) both died of infection by 2 weeks after viral challenge (P = .5) (data not shown). That this experiment was repeated and had similar results suggests that siRNA W86 failed to protect mice from West Nile virus encephalitis when administered 24 h after viral challenge.
DISCUSSION
The results of the present study demonstrate that an siRNA administered to mice before challenge with West Nile virus affords partial protection from death. These results may potentially be altered by increasing or decreasing the viral dose or the amount of siRNA administered, as has been noted in passive immunization studies on E protein antisera [29]. Because this murine model of infection mimics human infection and because 10% of patients develop serious complications from infection with West Nile virus, a dose of virus (80 pfu) that causes death in the majority of mice was used in the present study. Although siRNA W86 inhibited viral replication in both Vero cells and mice, the other siRNAs that were tested were not as effective. The reasons for these differences may be related to the primary and secondary structures of the siRNA, the targeted viral RNA, and the viral or cellular proteins that bind to the RNA and prevent recognition of the target by the RISC [35]. These data therefore suggest that specific siRNAs may have utility as a means of combating West Nile virus.
The lethal effect of West Nile virus in mice is related, in part, to the viral load. The virus disseminates in mice over the course of several days, enters the brain, and replicates to a high viral load at 1 week after infection, at which time the mice begin to die [3638]. A decrease in viral dose causes a delay in the kinetics of viral penetration and replication in the brain and therefore delays death [39]. In the present study, when siRNA W86 was given before viral challenge, viral replication in the liver was inhibited, and there was a subsequent diminished viral load in the brain, which thus delayed death or protected mice from death. siRNA W86 failed, however, to protect mice from death when it was injected 24 h after infection with West Nile virus. It is possible that, at 24 h after infection, the virus had already replicated enough to dampen the effects of the administration of siRNA at this point or that hydrodynamic injection, in which a large volume of fluid is injected into the mice, may have altered viral distribution and dissemination. These data, nevertheless, show that siRNA W86 had an effect on the outcome of West Nile virus infection in vivo.
Although hydrodynamic injection is useful in experimental studies, it is not appropriate for therapeutic delivery to patients. The hydrodynamic method has been used to deliver a synthetic siRNA or DNA encoding a short hairpin RNA into the murine portal or tail vein, and this method resulted in the effective blocking of the expression of HCV [10], HBV [40], and the fas [8] gene. The process of injecting such a large volume of solution (10% of the total body weight) into a murine vein cannot be applied to humans. Moreover, transfection of synthetic siRNA fragments only transiently inhibits virus replication. The development of viral vectors such as retrovirus, lentivirus, and adeno-associated virus to deliver a short hairpin RNAexpressing cassette can provide a more sustained inhibition of HIV [14, 15, 41], HCV [17, 18], HBV [40], influenza virus [42], or dengue virus [43] in mammalian cells. However, the safety of these viral delivery systems must be demonstrated before their adoption for clinical use. In addition, the effectiveness of RNAi relies on matched nucleotides in the targeted region of the gene, and a single-base nucleotide mismatch can abrogate activity [44], as has been observed with poliovirus and HIV [4547]. The development of better delivery systems and the assessment of diverse targeted siRNAs, either alone or in combination, will be required for successful siRNA therapy in humans.
West Nile virus continues to spread throughout North America, and effective therapy is lacking. In the present study, we have provided several lines of evidence that siRNAs can inhibit West Nile virus replication in vitro and in vivo and can alter the course of infection in mice. West Nile virus infection in humans is highly variable, ranging from asymptomatic infection to lethal encephalitis, and therefore it is only partially mimicked in the murine model, in which the onset of disease is rapid and the outcome is uniformly lethal. It is therefore possible that the effective administration of siRNA to patients early during the course of infection may reduce the viral load to a level at which the host immune response can effectively clear the virus and prevent disease. Although much work remains to be done to establish an effective delivery system for siRNA and to investigate other experimental models of West Nile virus, these data demonstrate that RNAi is a potential strategy to combat West Nile encephalitis in vivo.
Acknowledgments
We thank Debby Beck, for technical assistance, and Bao-Zhu Yang, for help with the statistical analysis.
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