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Antiviral and neuroprotective role of octaguanidinium dendrimer-conjugated morpholino oligomers in Japanese encephalitis.

Nazmi A, Dutta K, Basu A - PLoS Negl Trop Dis (2010)

Bottom Line: Plaque assay and immunoblot analysis performed from brain homogenates showed reduced viral load and viral protein expression, resulting in greater survival of infected animals.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.

View Article: PubMed Central - PubMed

Affiliation: National Brain Research Centre, Manesar, Haryana, India.

ABSTRACT

Background: 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.

Methodology/principal findings: 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. In vitro studies also showed that there was decrease in infective viral particle production following Morpholino treatment.

Conclusions/significance: 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.

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Related in: MedlinePlus

MOs treatment reduces the viral load in vivo.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) (A). Immunoblot analysis showed expression of JEV NS5 and E glycoprotein were significantly increased in JEV-infected and JEV+SC-MO than Sham, but reduced significantly after both 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) (B–D). Immunostaining of brain sections from different treatment groups showed greater presence of JEV antigen in JEV and JEV+SC-MO groups (E). Magnification ×20; scale bar correspond to 50µ Photomicrographs shown here in this figure are representative of three individual animals from each group.
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pntd-0000892-g003: MOs treatment reduces the viral load in vivo.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) (A). Immunoblot analysis showed expression of JEV NS5 and E glycoprotein were significantly increased in JEV-infected and JEV+SC-MO than Sham, but reduced significantly after both 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) (B–D). Immunostaining of brain sections from different treatment groups showed greater presence of JEV antigen in JEV and JEV+SC-MO groups (E). Magnification ×20; scale bar correspond to 50µ Photomicrographs shown here in this figure are representative of three individual animals from each group.

Mentions: 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 materials and methods section. Number of PFU/mL of the brain homogenate was found to be significantly higher in both JEV and JEV+SC-MO groups when compared to Sham (p<0.001). Viral PFUs were found to be significantly reduced following 3′ MO and 5′ MO treatment when compared to only JEV-infected or JEV+SC-MO group (p<0.001) (Figure 3A).


Antiviral and neuroprotective role of octaguanidinium dendrimer-conjugated morpholino oligomers in Japanese encephalitis.

Nazmi A, Dutta K, Basu A - PLoS Negl Trop Dis (2010)

MOs treatment reduces the viral load in vivo.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) (A). Immunoblot analysis showed expression of JEV NS5 and E glycoprotein were significantly increased in JEV-infected and JEV+SC-MO than Sham, but reduced significantly after both 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) (B–D). Immunostaining of brain sections from different treatment groups showed greater presence of JEV antigen in JEV and JEV+SC-MO groups (E). Magnification ×20; scale bar correspond to 50µ Photomicrographs shown here in this figure are representative of three individual animals from each group.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990691&req=5

pntd-0000892-g003: MOs treatment reduces the viral load in vivo.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) (A). Immunoblot analysis showed expression of JEV NS5 and E glycoprotein were significantly increased in JEV-infected and JEV+SC-MO than Sham, but reduced significantly after both 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) (B–D). Immunostaining of brain sections from different treatment groups showed greater presence of JEV antigen in JEV and JEV+SC-MO groups (E). Magnification ×20; scale bar correspond to 50µ Photomicrographs shown here in this figure are representative of three individual animals from each group.
Mentions: 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 materials and methods section. Number of PFU/mL of the brain homogenate was found to be significantly higher in both JEV and JEV+SC-MO groups when compared to Sham (p<0.001). Viral PFUs were found to be significantly reduced following 3′ MO and 5′ MO treatment when compared to only JEV-infected or JEV+SC-MO group (p<0.001) (Figure 3A).

Bottom Line: Plaque assay and immunoblot analysis performed from brain homogenates showed reduced viral load and viral protein expression, resulting in greater survival of infected animals.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.

View Article: PubMed Central - PubMed

Affiliation: National Brain Research Centre, Manesar, Haryana, India.

ABSTRACT

Background: 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.

Methodology/principal findings: 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. In vitro studies also showed that there was decrease in infective viral particle production following Morpholino treatment.

Conclusions/significance: 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.

Show MeSH
Related in: MedlinePlus