Japanese encephalitis virus (JEV) infection induces uncontrolled neuronal apoptosis, leading to irreversible brain damage. However, the mechanism of JEV-induced neuronal apoptosis has not been clearly elucidated. This study aimed to investigate both virus replication and neuronal cell apoptosis during JEV infection in human neuroblastoma SH-SY5Y cells. As a result, the kinetic productions of new viral progeny were time- and dose-dependent. The stimulation of SH-SY5Y cell apoptosis was dependent on the multiplicity of infections (MOIs) and infection periods, particularly during the late period of infection. Interestingly, we observed that of full-length Bax (p21 Bax) level started to decrease, which corresponded to the increased level of its cleaved form (p18 Bax). The formation of p18 Bax resulting in cytochrome c release into the cytosol appeared to correlate with JEV-induced apoptotic cell death together with the activation of caspase-3/7 activity, especially during the late stage of a robust viral infection. Therefore, our results suggest another possible mechanism of JEV-induced apoptotic cell death via the induction of the proteolysis of endogenous p21 Bax to generate p18 Bax. This finding could be a new avenue to facilitate novel drug discovery for the further development of therapeutic treatments that could relieve neuronal damage from JEV infection.

Zika virus (ZIKV) is an emerging flavivirus that is causing significant public health concerns worldwide because of its association with severe neurological disorders in both newborns and adults. At present, the search for effective antiviral drugs for ZIKV infection is intense. Melatonin is a molecule that influences a wide variety of physiological processes. Melatonin is a direct free radical scavenger, an indirect antioxidant due to its stimulation of antioxidant enzymes and an anti-inflammatory and immunoregulatory molecule; recent studies also have shown melatonin to have anti-viral activity. Here, we are the first to show that melatonin inhibits ZIKV replication in both Vero cells and SK-N-SH cells. The suppressive actions of melatonin pretreatment on ZIKV infection was found to be time- and dose-dependent. The inhibitory effect of melatonin was more pronounced in human SK-N-SH neural cells than in Vero cells. Molecular docking experiments showed that melatonin exhibited high binding affinity to ZIKV nonstructural 3 (NS3) protein, the possible underlying inhibitory mechanism of melatonin on ZIKV replication. The results suggest that melatonin may have potential prophylactic and treatment effects against ZIKV infection.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes high mortality rates in humans and it is the most clinically important and common cause of viral encephalitis in Asia. To date, there is no specific treatment for JEV infection. Melatonin, a neurotropic hormone, is reported to be effective in combating various bacterial and viral infections. However, the effects of melatonin on JEV infection have not yet been studied. The investigation tested the antiviral effects of melatonin against JEV infection and elucidated the possible molecular mechanisms of inhibition. Melatonin inhibited the viral production in JEV-infected SH-SY5Y cells in a time- and dose-dependent manner. Time-of-addition assays demonstrated a potent inhibitory effect of melatonin at the post-entry stage of viral replication. Molecular docking analysis revealed that melatonin negatively affected viral replication by interfering with physiological function and/or enzymatic activity of both JEV nonstructural 3 (NS3) and NS5 protein, suggesting a possible underlying mechanism of JEV replication inhibition. Moreover, treatment with melatonin reduced neuronal apoptosis and inhibited neuroinflammation induced by JEV infection. The present findings reveal a new property of melatonin as a potential molecule for the further development of anti-JEV agents and treatment of JEV infection.

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