Conceived and designed the experiments: AN KD AB. Performed the experiments: AN KD. Analyzed the data: AN KD AB. Wrote the paper: AN KD AB.
The authors have declared that no competing interests exist.
Japanese encephalitis (JE), caused by a mosquito-borne flavivirus, is endemic to the entire south-east Asian and adjoining regions. Currently no therapeutic interventions are available for JE, thereby making it one of the most dreaded encephalitides in the world. An effective way to counter the virus would be to inhibit viral replication by using anti-sense molecules directed against the viral genome. Octaguanidinium dendrimer-conjugated Morpholino (or Vivo-Morpholino) are uncharged anti-sense oligomers that can enter cells of living organisms by endocytosis and subsequently escape from endosomes into the cytosol/nuclear compartment of cells. We hypothesize that Vivo-Morpholinos generated against specific regions of 3′ or 5′ untranslated regions of JEV genome, when administered in an experimental model of JE, will have significant antiviral and neuroprotective effect.
Mice were infected with JEV (GP78 strain) followed by intraperitoneal administration of Morpholinos (5 mg/kg body weight) daily for up to five treatments. Survivability of the animals was monitored for 15 days (or until death) following which they were sacrificed and their brains were processed either for immunohistochemical staining or protein extraction. Plaque assay and immunoblot analysis performed from brain homogenates showed reduced viral load and viral protein expression, resulting in greater survival of infected animals. Neuroprotective effect was observed by thionin staining of brain sections. Cytokine bead array showed reduction in the levels of proinflammatory cytokines in brain following Morpholino treatment, which were elevated after infection. This corresponded to reduced microglial activation in brain. Oxidative stress was reduced and certain stress-related signaling molecules were found to be positively modulated following Morpholino treatment.
Administration of Vivo-Morpholino effectively resulted in increased survival of animals and neuroprotection in a murine model of JE. Hence, these oligomers represent a potential antiviral agent that merits further evaluation.
Japanese encephalitis (JE) is caused by a flavivirus that is transmitted to humans by mosquitoes belonging to the
The genus
The JEV genome is approximately 11 kb in length that carries a single long open reading frame (ORF) flanked by a 95-neucleotide 5′ untranslated region (5′ UTR) and a 585-neucleotide 3′ UTR. The ORF encodes a polyprotein which is processed by viral and cellular proteases into three structural and seven non structural proteins
Anti-sense oligonucleotides have been shown to be effectively used as therapeutic agents against viral infection. In one such study siRNA generated against 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) has been found to protect against lethal encephalitis
Morpholino oligomers are single stranded DNA analogues containing same nitrogenous bases as DNA but joined by backbone consisting of morpholine rings and phosphorodiamidate linkages
All animal experiments were approved by the institutional animal ethical review board named “Institutional Animal and Ethics Committee of National Brain Research Centre”. The animal experiment protocol approval no. is NBRC/IAEC/2007/36. Animals were handled in strict accordance with good animal practice as defined by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forestry, Government of India.
Vero cells (a kind gift from Dr. Guruprasad Medigeshi, Translational Health Science and Technology Institute, Gurgaon, India) and Neuro2A (obtained from National Centre for Cell Science, Pune, India) cells were grown in DMEM (Dulbecco's modified Eagles medium, supplemented with 10% fetal bovine serum (FBS) and antibiotics. The GP78 strain of JEV was propagated in suckling BALB/c mice and their brains were harvested when symptoms of sickness were observed. A 10% tissue suspension was made in MEM (minimum essential medium), followed by centrifugation at 10,000× g and finally filtered through a 0.22 µ sterile filter
All Vivo-Morpholino (MO) oligos were commercially procured from Gene Tools LLC, (Philomath, OR, USA). MOs were designed to be complementary to sequences in the JEV (GP78 strain) genome, as shown in
Schematic diagram showing the secondary structure of the 3′ and 5′ UTR of JEV genomic RNA as predicted by the Mfold program
Morpholino name | Morpholino sequence(5′-3′) | Targeted nucleotides in the JEV genome(GenBank: AF075723.1) |
3′ MO | 10861–10882 | |
5′ MO | 9–22 | |
SC-MO | NA |
NA- Not applicable.
Five to six weeks old BALB/c mice of either sex were randomly distributed into 5 groups- Sham, JEV-infected, JEV-infected and treated with scrambled Morpholino (JEV+SC-MO), JEV-infected and treated with Morpholino against viral 3′ conserved region (JEV+3′ MO) and JEV-infected and treated with Morpholino against secondary structure in the 5′UTR of viral RNA (JEV+5′ MO). Initially each group contained 8 animals. Animals belonging to all groups except Sham were infected with 3×105 plaque forming units (PFU) of JEV (GP78 strain) and that day was considered as day zero
Mouse cytokine bead array (CBA) kits were used to quantitatively measure cytokine levels in mouse whole-brain lysates. 50 µL of bead mix containing a population of beads with distinct fluorescence intensities that have been coated with capture antibodies for different cytokines, and 50 µL of whole-brain lysates were incubated together, along with equal volume of phycoerythrin (PE)-conjugated detection antibodies, for 2 h at room temperature, in dark. The beads were then washed and re-suspended in 300 µL of supplied 1× wash buffer. The beads were acquired using Cell Quest Pro Software in FACS Calibur and analyzed using BD CBA software (Becton Dickinson, San Diego, CA). Standard curve was prepared by incubating 50 µL of supplied mouse inflammation standards with 50 µL of bead mix and PE-conjugated detection antibodies
Protein concentrations of whole brain lysates were estimated by Bradford method. Sample volumes containing 20 µg of protein were electrophoresed on polyacrylamide gel and transferred onto nitrocellulose membrane. After blocking with 7% skimmed milk, the blots were incubated overnight at 4°C with primary antibodies against JEV E-glycoprotein (Abcam, USA), and JEV NS5 (a kind gift from Dr. Chun-Jung Chen, Taichung Veterans General Hospital, Taichung, Taiwan), iNOS (Upstate-Chemicon, USA), HSP-70, SOD-1 (Santa Cruz Biotechnology, CA, USA), TRX (AB Frontiers, Korea; a kind gift from Dr. Ellora Sen, NBRC), phospho NFκB, phospho ERK1/2, total ERK1/2 and phosphoP38 MAP kinase (Cell Signaling, USA) at 1∶1000 dilutions. After extensive washes with PBS–Tween, blots were incubated with appropriate secondary antibodies conjugated with peroxidase (Vector Laboratories, CA, USA). The blots were again washed with PBS–Tween and processed for development using chemiluminescence reagent (Millipore, USA). The images were captured and analyzed using Chemigenius, Bioimaging System (Syngene, Cambridge, UK). The blots were stripped and reprobed with anti-β-tubulin (Santa Cruz Biotechnology, USA) to determine equivalent loading of samples
For immunohistochemical staining, brains from scarified animals were excised following repeated transcardial perfusion with ice-cold saline and fixed with 4% paraformaldehyde. Twenty micron thick cryosections were made with the help of Leica CM3050S cryostat and processed for immunohistochemical staining to detect presence of JEV antigen in the brain and to label activated microglia. Sections were incubated overnight at 4°C with mouse anti-JEV antigen (Nakayama, 1∶250) (Chemicon, CA, USA) and rabbit anti-Iba-1 (1∶ 500; Wako, Osaka, Japan), respectively. After washes, slides were incubated with FITC-conjugated anti-mouse or anti-rabbit secondary antibodies (Vector laboratories Inc. Burlingame, USA) and following final washes, sections were sections were cover slipped after mounting with 4′-6-diamidino-2-phenylindole (DAPI, Vector laboratories Inc.). The slides were observed under Zeiss Axioplan 2 fluorescence microscope and Zeiss Apotome microscope (Zeiss, Gottingen, Germany), respectively
Cryosections of brain from Sham-treated, JEV-infected and JEV-infected and MO treated animals were rinsed in de-ionized water followed by incubation with the thionin dye. The excess dye was washed off and the slides were immersed in alcohol-dioxane (1∶1) solution for differentiation. After two changes in xylene the slides were mounted with DPX and observed under a Leica 4000 DB light microscope (Leica Microsystems, USA)
The level of ROS produced within brain tissue of each treatment groups were measured by the cell permeable, non-polar, H2O2-sensitive probe 5(and 6)-chlromethyl-20,70-dichlorodihydrofluoresceindiacetate (CM-H2DCFDA; Sigma, USA). CM-H2DCFDA diffuses into cells, where its acetate groups are cleaved by intracellular esterases, releasing the corresponding dichlorodihydrofluorescein derivative. Subsequent oxidations of CM-H2DCFDA yields a fluorescent adduct dichlorofluorescein that is trapped inside the cell. Brain homogenates were treated with 5 µM solution of CM-H2DCFDA followed by incubation in dark at room temperature for 45 min and then the relative fluorescence intensity were measured with the help of Varioskan Flash multimode reader (Thermo Electron, Finland) at excitation 500 nm and emission 530 nm. The fluorescence intensity of intracellular CM-H2DCFDA is a linear indicator of the amount of H2O2 in the cells. The measured mean fluorescence intensity was then normalized to equal concentrations of protein in each sample
Nitric oxide released from brain homogenates following MO treatment was assessed using Griess reagent as described previously. Briefly, 100 µL of Griess reagent (Sigma, St. Louis, USA) was added to 100 µL of brain homogenate and incubated in dark for 15 min. The intensity of the color developed was estimated at 540 nm with the help of a Benchmark plus 96-well ELISA plate reader (Biorad, CA, USA). The amount of nitrite accumulated was calculated (in µM) from a standard curve constructed with different concentrations of sodium nitrite
Mouse neuroblastoma cells (N2a) were plated in five 60 mm plates at a density of 5×105 cells/plate, and were cultured for 18 h. After 6 h in serum free DMEM, cells were either mock-infected with sterile 1× PBS or infected with JEV at multiplicity of infection (MOI) of 5. After 1½ h, cells were washed twice with sterile 1× PBS to remove non-internalized virus. Three of the four plates that were infected with JEV, were treated with SC-MO, 3′ MO and 5′ MO at 10 µM concentrations and all plates were incubated for 24 h in serum free media.
After two washes with 1× PBS, cells were first fixed with BD cytofix solution (BD Biosciences) for 15 min and permeabilized by resuspending in permeabilization buffer (BD Cytoperm plus; BD Biosciences) and incubated at 25°C for at least 10 min. Cells were then washed twice in wash buffer (PBS containing 1% bovine serum albumin) then resuspended in wash buffer at 1×106 cells per 100 µL. Primary antibody (JEV Nakayama strain; Chemicon, USA) were added in 1∶100 dilutions and incubated for 30 min at 25°C. The cells were washed with wash buffer and pelleted by centrifugation followed by incubation with FITC conjugated secondary antibody for 30 min. After final wash with wash buffer, cells were resuspended in 400 µL FACS buffer and analyzed on a FACS Calibur. The percentage of population of JEV-positive cells was calculated after gating the populations on a Dot plot using Cell Quest Pro Software (BD Biosciences).
Statistical analysis was performed using SIGMASTAT software (SPSS Inc., Chicago, IL, USA). Data were compared between groups using one-way analysis of variance followed by post hoc test. Differences upto p<0.05 were considered significant.
MO treatment conferred significant protection to mice following JEV infection. The survival of mice following JEV infection was dramatically increased with treatments of both 3′ and 5′ MO. Approximately 90% of all the animals that were treated with 3′ MO survived as compared to 75% survival of those animals that were treated with 5′MO, post infection with JEV
The survival of mice following JEV infection was dramatically increased with treatments of both 3′ and 5′ MO, though the survival in 3′ MO treated mice was greater (∼90%) than those treated with 5′MO (75%) (A). Considerable changes in the average body weights of JEV-infected animals treated with both 3′ and 5′ MO were not observed when compared to animals belonging to JEV and JEV+ SC-MO groups that showed significant reductions in their body weights from 6th day post infection till their death. Black arrows points to the days by which all the animals died. (B). Infection with JEV was accompanied with distinct symptoms that were alleviated following treatments with both 3′ and 5′ MO. Animals were assigned scores according to the symptoms, in a blinded manner. The graph was plotted by taking the scores of one animal that was considered as the representative of that group (C). n = 8 for all experiments; data shown are representative of duplicate sets of experiments.
To assess whether the MOs has any effect on reduction of viral load in brain, homogenized brain samples from all the treatment groups were subjected to plaque assay as described in
Significant reduction in viral titer was observed following MO treatment in animals, as compared to JEV-infected and JEV+SC-MO groups. (* p<0.001 for JEV and JEV+SC-MO when compared to Sham, # p<0.001 for JEV+ 3′MO and JEV+ 5′ MO when compared to only JEV-infected group) (
To further validate the results obtained from the plaque assay, immunoblot for some of the JEV-specific proteins were performed. The expression of NS5, a non structural protein of JEV, was significantly increased in JEV and JEV+SC-MO groups when compared to Sham (p<0.01), but its level were found to be significantly reduced after both 3′ and 5′ MO treatments when compared to JEV-infected group (p<0.01). Similarly, E glycoprotein level showed significant increase in JEV-infected and JEV+SC-MO groups when compared to Sham (p<0.01) which were then drastically reduced following 3′ and 5′ MO treatments (p<0.01)
To further characterize the inhibitory effects of MO on JEV-induced neuronal death, brain sections from all the treatment groups were subjected to thionin staining. Numerous healthy cells were seen in sections obtained from Sham, JEV+3′ MO and JEV+5′ MO groups when compared to sections belonging to only JEV-infected or JEV+SC-MO groups which contained numerous unhealthy/dying neurons with altered morphology
Thionin staining of brain sections from all the treatment groups showed neurons with distinct morphology in Sham-treated, JEV+3′ MO and JEV+5′ MO groups. However in sections from JEV-infected and JEV+SC-MO groups showed damaged neurons with altered morphology. Magnification ×20; scale bar correspond to 50 µ. Immunofluorescent staining for microglia-specific Iba-1 performed in brain sections of all groups showed that number of activated (star shaped) microglia appeared to be more frequent in JEV-infected and JEV+SC-MO groups as compared to compared to sections belonging to Sham, JEV+3′ MO and JEV+5′ MO groups. Magnification ×20; scale bar correspond to 50 µ (B). Photomicrographs shown here in this figure are representative of three individual animals from each group. CBA showed levels of MCP-1, IFN-γ, TNF-α, and IL-6 were increased significantly in both JEV-infected and JEV+SC-MO groups when compared to Sham treated groups. The elevated levels of these proinflammatory cytokines were then significantly reduced with 3′ and 5′ MO treatments (* p<0.01 for JEV and JEV+SC-MO when compared to Sham; # p<0.01 for JEV+ 3′MO and JEV+ 5′ MO when compared to only JEV-infected group) (C–F).
Microglial activation and increased proinflammatory cytokine production are the hallmarks of JEV infection
Increased oxidative stress in CNS is a major outcome of JEV infection
Increases were observed in the ROS levels in the brain samples of JEV-infected and JEV+SC-MO groups in comparison to Sham, that were reduced following 3′ and 5′ MO treatments. Although ROS levels were decreased in JEV+3′ MO group when compared to JEV-infected group, it remained significantly higher than that in Sham (A). Approximately 13-fold increases were observed in the levels of HSP-70 in JEV-infected and JEV+SC-MO groups as compared to Sham. These drastic increases were significantly reduced in JEV+3′ MO and JEV+5′ MO groups, the levels remained significantly higher than Sham (B&C). SOD-1 levels were found to be elevated 2- and nearly 3-fold in JEV-infected and JEV+SC-MO groups respectively compared to Sham. 3′ and 5′ MO treatment caused significant reduction of SOD-1 levels compared to JEV-infected group (B&D). Alterations in TRX-1 levels were similar to that observed in SOD-1 except that its level in JEV+ SC-MO was not significantly different than only JEV-infected group (B&E). Nearly 2-fold increases were observed in NO levels of JEV-infected and JEV+SC-MO groups when compared to those obtained from Sham. NO levels were subsequently reduced to significantly lower levels following 3′ and 5′MO treatments (F). iNOS expression was found to increase 8-fold in JEV-infected and JEV+SC-MO groups when compared to Sham. Following 3′ and 5′MO treatments iNOS levels decreased significantly as compared to JEV-infected group (G&H). (* p<0.01 for JEV when compared to Sham; ** p<0.01 for JEV+SC-MO when compared to JEV-infected only; # p<0.01 for JEV+ 3′MO and JEV+ 5′ MO when compared to only JEV-infected group; ̂ p<0.01 for JEV+ 3′MO when compared to Sham; $ p<0.01 for JEV+ 5′ MO when compared to Sham).
JEV infection leads to increased nitric oxide (NO) production in CNS
Western blot analysis demonstrated a significant inhibition in the expression of different stress related proteins whose levels were elevated following JEV infection. Upon MO treatments there were approximately 4-fold increases in the levels of pNFκB in JEV and JEV+SC-MO groups when compared to Sham (p<0.01). The levels of pNFκB were found to be significantly reduced in JEV+3′ MO and JEV+5′ MO groups when compared to only JEV-infected groups (p<0.01)
Approximately 4-fold increases in the levels of pNFκB and 3-fold increases in the levels of phosphoP38MAPK in JEV-infected and JEV+SC-MO groups were observed in comparison to Sham. The levels of both pNFκB and phosphoP38MAPK were significantly reduced in JEV+3′ MO and JEV+5′ MO groups when compared to only JEV-infected groups (A–C). Phospho ERK1 and ERK2 levels were found to be increased by approximately 3- and 5-fold in JEV-infected and JEV+SC-MO groups respectively, when compared to Sham. but showed considerable decreases in JEV+3′ MO and JEV+5′ MO groups when compared to only JEV-infected groups (A&D) (* p<0.01 for JEV-infected and JEV+SC-MO when compared to Sham; # p<0.01 for JEV+3′ MO and JEV+5′MO when compared to only JEV-infected groups).
To assess whether MO has any effect on viral load
Use of anti-sense molecules for targeted inhibition of viral replication has been under investigation for quite sometime. Though the application of these molecules has raised the possibilities of their future use as novel therapeutic agents, there are many issues regarding their effectiveness in terms of their stability and delivery to targeted cells. Recent studies are involved in developing techniques to minimize or eliminate these issues so that anti-sense therapy can be employed to a wide variety of intractable diseases such as splice-modifying genetic defects and viral diseases. The role of various anti-sense molecules in the inhibition of replication of JEV has been reported with positive outcomes
Morpholino oligomers are single stranded anti-sense molecules that exert their action by steric blocking of complementary RNA. Unlike other types of anti-sense oligonucleotides, Morpholinos provide all the desired properties of stability, nuclease resistance, high efficacy, long-term activity, water solubility, low toxicity, and exquisite specificity. Morpholino oligomers has been used previously for the inhibition of flaviviral replication
To minimize the problems encountered by the peptide-conjugated Morpholinos, octaguanidinium dendrimer-conjugated Morpholino oligomers have been developed that are commonly referred to as Vivo-Morpholino (MO). These custom-sequence anti-sense molecules have been reported to enable Morpholino applications in adult animals. MO was our choice of anti-sense molecule as this enabled us to test the specifically designed oligonucleotides in both animal as well as cell culture models. Though ‘outstanding’ results have been reported to be achieved by intravenous (i.v.) administration of the MO, we preferred the intraperitoneal route via which modest systemic delivery can be achieved. This was so done because brain has been reported to be an ineffective tissue when MOs are administered i.v.
Plaque assay from the brain homogenates of animals of all groups revealed that the number of infective viral particle production was dramatically reduced following 3′ and 5′ MO treatment. The 3′ MO was generated against the 3′ CSI region of the JEV genome that interacts with 5′ CS region located in coding sequence for capsid protein at 136–146 nucleotides from 5′ terminal of the genome. This interaction results in cyclization of JEV genome that is necessary for its efficient replication. The 5′ MO was targeted towards one of the secondary structures of the 5′ UTR that are required for the formation of translation pre-initiation complex. Blocking of these two sites in the JEV genome leads to the most likely effect, i.e. inhibition of replication and translation of viral genome. This was further corroborated by the decrease in the expressions of viral proteins (NS5, E glycoprotein and general flaviviral envelop protein) in the brain. Flaviviral NS5 is known to possess guanylyltransferase activity that helps in the synthesis of methylated cap structure at the 5′ end of the viral genome that plays a crucial role in the translation and stability of mRNAs
It is well known that JEV infection causes microglial activation. Activated microglia releases an array of chemical mediators that are detrimental for the neurons in brain
Generation of ROS with the generation of oxidative damage has been implicated in neurodegenerative diseases and in the degradation of nervous system functions and are also reported to increase following JEV infection
Activation of pNFκB regulates apoptotic genes, especially the TRAF1 and TRAF2, and thereby checks the activities of the caspases, which are central to most apoptotic processes. JEV is known to activate pNFκβ via a PI3K-dependent pathway in the brain of infected animals, which is associated with apoptosis
To confirm the anti-viral and neuroprotective property of the MOs observed in
This study was undertaken to determine the antiviral and neuroprotective efficacy of Vivo-Morpholinos in an experimental model of JE so that it can be considered as a therapeutic agent in the near future. There have been studies regarding the anti-JEV effects of other types of Morpholino oligomers though none of them are yet to be considered for therapeutic purposes. This is the first study that investigates the role of Morpholino oligomers specially designed for effective delivery into live animal models. Generally, the i.p route of administration of any drug is preferred in animal studies over any other routes. However, the efficacy of these antisense molecules needs to be checked by administering through other applicable routes, as i.p. administration in humans is uncommon, though not unheard of. The amounts of oligomers required and the route of administration in this study marks these molecules as practicable therapeutic agents in JE, though further studies are required before these can be recommended for clinical trials.
MO treatments decrease viral load
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The authors would like to thank Sulagna Das and Prosenjit Pal for their help; Kanhaiya Lal Kumawat and Manish Kumar Dogra for their technical assistance.