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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

Mice are protected from JEV following MO treatment.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.
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pntd-0000892-g002: Mice are protected from JEV following MO treatment.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.

Mentions: 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 (Figure 2A). Infection with JEV was accompanied with distinct symptoms and weight loss whereas treatments with both 3′ and 5′ MO post JEV infection, prevented animals from suffering. Not much considerable changes in the average body weights of JEV-infected animals treated with both 3′ and 5′ MO were observed when compared to animals belonging to JEV and JEV+ SC-MO groups showing significant reductions in their body weights 6 days post infection (Figure 2B). The symptoms associated with JE in murine model were observed on daily basis and scores were attributed accordingly. The animals that had most symptoms received the highest scores. It was observed that 3′ and 5′ MO treated animals scored lesser than those belonging to the JEV-infected or JEV+SC-MO groups (Figure 2C).


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

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

Mice are protected from JEV following MO treatment.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.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0000892-g002: Mice are protected from JEV following MO treatment.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.
Mentions: 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 (Figure 2A). Infection with JEV was accompanied with distinct symptoms and weight loss whereas treatments with both 3′ and 5′ MO post JEV infection, prevented animals from suffering. Not much considerable changes in the average body weights of JEV-infected animals treated with both 3′ and 5′ MO were observed when compared to animals belonging to JEV and JEV+ SC-MO groups showing significant reductions in their body weights 6 days post infection (Figure 2B). The symptoms associated with JE in murine model were observed on daily basis and scores were attributed accordingly. The animals that had most symptoms received the highest scores. It was observed that 3′ and 5′ MO treated animals scored lesser than those belonging to the JEV-infected or JEV+SC-MO groups (Figure 2C).

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