Plaque assay from the brain homogenates of animals of all groups revealed that the number of infective viral particle production was dramatically reduced following 39 and 59 MO treatment. The 39 MO was generated against the 39 CSI region of the JEV genome that interacts with 59 CS region located in coding sequence for capsid protein at 136-146 nucleotides from 59 terminal of the genome. This interaction results in cyclization of JEV genome that is necessary for its efficient replication. The 59 MO was targeted towards one of the secondary structures of the 59 UTR that are required for the formation of translation preinitiation 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 Vivo-Morpholino in Japanese Encephalitis www.plosntds.org 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 59 end of the viral genome that plays a crucial role in the translation and stability of mRNAs [34] . The JEV E glycoprotein is believed to be involved in viral adhesion and entry into host cells, hemagglutination, cellular tropism, viral virulence, and the induction of protective immune responses [35] . Decreased expression of these proteins indicates that viral replication and production of new infective viral particles are inhibited due to the MOs. Immunohistochemical staining for viral antigen also provided visual confirmation of the fact that JEV antigen was detected at much lower amounts in the brain following MO treatment. However, these data does not prove that MOs directly inhibit infective viral particle production in the brain itself, as it cannot be conclusively stated whether the MOs can reach brain. These data merely suggests that the number of replication-competent infective JEV in the brain was significantly reduced, which subsequently leads to neuroprotection.