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. 2006 Apr;3(4):e96.
doi: 10.1371/journal.pmed.0030096. Epub 2006 Feb 14.

A single siRNA suppresses fatal encephalitis induced by two different flaviviruses

Priti Kumar  1 , Sang Kyung Lee, Premlata Shankar, N Manjunath
Affiliations

A single siRNA suppresses fatal encephalitis induced by two different flaviviruses

Priti Kumar et al. PLoS Med. 2006 Apr.

Abstract

Background: Japanese encephalitis virus (JEV) and West Nile virus (WNV) are neurotropic flaviviruses that can cause acute encephalitis with a high fatality rate. Currently there is no effective treatment for these infections.

Methods and findings: We tested RNA interference (RNAi)-based intervention to suppress lethal JE and WN encephalitis in mice. To induce RNAi, we used either lentivirally expressed short hairpin RNA (shRNA) or synthetic short interfering RNA (siRNA). As target, we selected the cd loop-coding sequence in domain II of the viral Envelope protein, which is highly conserved among all flaviviruses because of its essential role in membrane fusion. Using as a target a species-specific sequence in the cd loop that is conserved only among the different strains of either JEV or WNV, we could achieve specific protection against the corresponding virus. However, by targeting a cross-species conserved sequence within the cd loop, we were able to protect mice against encephalitis induced by both viruses. A single intracranial administration of lentivirally delivered shRNA or lipid-complexed siRNA before viral challenge or siRNA treatment after viral challenge was sufficient for protection against lethal encephalitis.

Conclusions: RNAi-based intervention affords near complete protection from both JEV- and WNV- induced encephalitis in mice. Our results show, to our knowledge for the first time, that siRNA can be used as a broad-spectrum antiviral agent for treating encephalitis caused by multiple related viruses.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Lentiviral Delivery of FvE J shRNA Suppresses JEV Replication in Neuro 2a Cell Line
(A) Northern blot shows intracellular processing of FvE J shRNA. RNA extracted from Neuro 2a cells stably transduced with shFvE J or shLuc lentivirus was probed with 32P end-labeled synthetic FvE J siRNA sense strand to detect intracellular processing of shRNA. Antisense strand of the synthetic FvE J siRNA (siFvE J) was used as positive control. Before loading, the samples were normalized for total RNA content. (B) shFvE J inhibits JEV replication in Neuro 2a cells. Mock- or lentivirally transduced Neuro 2a cells were challenged with JEV at a MOI of 1, and the viral replication monitored 60 h later by flow cytometry after staining the cells with a JEV envelope-specific antibody. Percent of infected cells is indicated. The results are representative of at least three independent experiments. (C) Titration of shFvE J-induced inhibition of JEV replication. Neuro 2a cells transduced with shFvE J or control shLuc lentivirus were challenged with the indicated MOIs of JEV, and viral replication was assessed by flow cytometry at different times postinfection. Percent inhibition of viral replication compared to mock-transduced cells is shown. Results are representative of three experiments. (D) shFvE J inhibits accumulation of JEV genomic RNA. Total RNA obtained from the control shLuc- or shFvE J lentivirus-transduced cells, which were either uninfected (UI) or infected with JEV, was probed with JEV- or β-actin cDNA in a Northern blot analysis.
Figure 2
Figure 2. shFvE J Protects Mice against JEV-Induced Encephalitis
(A) BALB/c mice (ten per group) were injected IC on days −4, −2, and 0 with 2 × 10 5 TU of either shFvE J or shLuc lentiviruses. On day 0, 30 min after injection of the third dose of lentivirus, they were injected at the same spot IC with four LD 50 of JEV, and the mice were monitored for survival over time. (B) Brain sections from shFvE J treated mice reveal no flavivirus-induced pathology. Representative photomicrographs of hematoxylin and eosin-stained horizontal brain sections obtained from mice treated with shFvE J or shLuc lentivirus and infected with JEV for 5 d are shown at indicated magnifications. (C) Lack of infectious virus in the brains of shFvE J-treated mice. Mice were injected with lentiviruses and challenged with JEV as in (A), and their brain homogenates, obtained 5 d after JEV challenge, were plaque-titrated on BHK21 cell monolayers. For shFvE J lentivirus, viral titers after a single (1×) as well as three (3×) administrations are shown. The viral titers are shown as log plaque-forming units per total brain. Each symbol represents an individual mouse. (D) Brains from shFvE J treated mice are free of infectious virus. Brain homogenates in (C) were pooled, 1, 10, or 50 μl of pooled homogenate were inoculated onto Neuro 2a cells, and the viral replication was monitored by flow cytometry 5 d later. (E) A single IC injection with shFvE J is sufficient for protection against JE encephalitis. Mice (five per group) were injected IC with 2 × 10 5 TU of shLuc or shFvE J, challenged 30 min later with indicated doses of JEV, and observed for survival over time.
Figure 3
Figure 3. siFvE J Also Protects against Fatal JEV Infection
(A) Transfection of Neuro 2a cells with i-Fect complexed siFvE J confers protection against JEV infection comparable to lipofectamine transfection. Neuro 2a cells were transfected with siRNA mixed with i-Fect or lipofectamine and after 2 d, they were challenged with JEV at a MOI of ten. Viral replication was monitored 72 h postinfection by flow cytometry. Also shown is an overlay histogram of uninfected Neuro 2a cells and JEV-infected Neuro 2a cells treated prior to infection with either i-Fect/siLuc, lipofectamine/siFvE J, or i-Fect/siFvE J as indicated. (B) i-Fect-complexed siFvE J protects mice from JEV infection when injected 30 min but not 6 h after infection. Mice (five per group) were injected IC with four LD 50 of JEV, and after 30 min or 6 h they were also injected at the same spot with 0.5 nmoles of either siLuc or siFvE J complexed with i-Fect and monitored for survival over time. (C) i-Fect complexed siFvE J reduces the level of viral replication in mouse brain when administered 6h post challenge. Mice were injected with siRNAs 6 h after JEV challenge and brain homogenates obtained 3 d later were titrated on BHK21 cell monolayers. Log plaque-forming units per brain is shown. Each symbol represents an individual mouse. (D) Transfection of Neuro 2a cells with JetSI/DOPE complexed siFvE J results in inhibition of JEV replication. Neuro 2a cells were treated with siFvE J or siLuc as in a using JetSI/DOPE instead of i-Fect to complex the siRNAs. Overlay histogram denotations are indicated. (E) siFvE J complexed with JetSI/DOPE protects mice against fatal encephalitis. Mice (ten per group) were injected IC with four LD 50 of JEV and were treated either with 3.2 nmoles siLuc complexed with JetSI/DOPE after 30 min or with JetSI/DOPE complexed with siFvE J after 30 min, 6 h, or 18 h after infection and monitored for survival over time. (F) shFvE J fails to protect against WNV-induced encephalitis. Mice (five per group) were injected with 2 × 10 5 TU of RV-G pseudotyped shLuc or shFvE J lentiviruses and challenged 30 min later with four LD 50 of WNV and monitored for survival over time. (G) siFvE W protects mice against lethal WNV-induced encephalitis. Mice (ten per group) were infected IC with four LD 50 of WNV, and 30 min or 6 h later they were also injected with 3.2 nmoles of either control siLuc or siFvE W complexed with JetSI/DOPE, and monitored for survival over time.
Figure 4
Figure 4. FvE JW Protects against Encephalitis Induced by Either JEV or WNV
(A) FvE JW siRNA inhibits both JEV and WNV infection in Neuro 2a cells. siRNA-treated Neuro 2a cells were challenged with a MOI of ten of either JEV (left) or WNV (right) and examined for viral replication by flow cytometry 72 h postinfection. Included is an overlay histogram of uninfected Neuro 2a cells and JEV- or WNV-infected Neuro 2a cells transfected prior to infection with either JetSI/DOPE/siLuc or JetSI/DOPE/siFvE JW as indicated. (B) FvE JW siRNA protects mice against both JEV and WNV-induced encephalitis. Mice were injected IC with four LD 50 of JEV (left) or WNV (right), and after 30 min or 6 h they were also injected at the same spot with 3.2 nmoles of either siLuc or FvE JW complexed with JetSI/DOPE and monitored for survival over time. Ten and five mice per group were used to test the effect of siRNA 30 min and 6 h postinfection, respectively.
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References

    1. Chambers TJ, Hahn CS, Galler R, Rice CM. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol. 1990;44:649–688. - PubMed
    1. Mackenzie JS, Gubler DJ, Petersen LR. Emerging flaviviruses: The spread and resurgence of Japanese encephalitis, West Nile and dengue viruses. Nat Med. 2004;10:S98–S109. - PubMed
    1. Seligman SJ, Gould EA. Live flavivirus vaccines: reasons for caution. Lancet. 2004;363:2073–2075. - PubMed
    1. Biggerstaff BJ, Petersen LR. Estimated risk of transmission of the West Nile virus through blood transfusion in the US, 2002. Transfusion. 2003;43:1007–1017. - PubMed
    1. Tyler KL. West Nile virus infection in the United States. Arch Neurol. 2004;61:1190–1195. - PubMed

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