Introduction: Japanese encephalitis virus (JEV) is the most principal cause of viral encephalitis in the Asia-Pacific region. Being neurotropic, the principal target cells for JEV are neurons. In addition to direct viral infection of neurons, bystander damage caused by pro-inflammatory cytokines released from activated microglia is also involved in JEV-induced neuronal death. MicroRNAs (miRNAs) are important in viral infections, since viruses affect cellular miRNA expression to control existing regulatory pathways to establish a host environment conducive to their replication. Hence, we sought to evaluate the possible role of miRNAs in JEV infection.
Methods: In vitro studies were performed in neuronal (HT22)/microglial (CHME3) cell line whereas JEV infected mouse was used as in vivo model. miRNA expression was assessed by qPCR and in situ hybridization. Whereas mimic and inhibitor were transfected to manipulate miRNA expressions in cells, Vivo-Morpholino was intracranially injected to inhibit miRNAs in mouse. .
Results: miR-301a was significantly increased in JEV infected neuron and microglia. In neuron, miR-301a repressed type I interferon by targeting interferon regulatory factor 1 (IRF1) and suppressor of cytokine signalling 5 (SOCS5) and neutralization of JEV-induced miR-301a reinforced host innate immunity by restoring IFN-β expression and restricting viral propagation. Moreover, in vivo inhibition of miR-301a rescued IRF1 and SOCS5, increased IFN-β response and reduced JEV replication. In JEV-infected microglia, miR-301a induced NF-κβ activation by targeting NF-κβ repressing factor (NKRF) and augmented inducible nitric oxide synthase, cyclooxygenase-2, and pro-inflammatory cytokine expression.
Conclusions: miR-301a inhibition not only induces type I interferon to strengthen neuronal innate immunity but also inhibits bystander damage of neurons by reducing microglial activation. Thus targeting miR-301a could provide a new insight to develop effective antiviral strategy.