25-Hydroxy cholesterol effectively inhibits Japanese encephalitis virus infection in a cellular model

The Japanese encephalitis virus (JEV) is a mosquito-borne, enveloped flavivirus that causes acute encephalitis. Although JEV is increasingly recognized as a global threat, there is currently no FDA-approved treatment available for JEV. 25-hydroxycholesterol (25-HC) is an oxysterol produced through the oxidation of cholesterol, a process catalyzed by cholesterol 25-hydroxylase (CH25H), which is an interferon-stimulated gene that is upregulated during viral infections. In this study, we report for the first time that 25-HC effectively prevents JEV infection in cells. Our results show that 25-HC inhibits JEV infection in Vero cells with an IC₅₀ of ∼4.18 μM, while displaying no cell toxicity (CC₅₀ > 50 μM). In addition, we show that the RNA level of the JEV envelope (E) protein exhibits a dose-dependent reduction in the presence of 25-HC, which is further supported by the concomitant decrease of the E protein expression. We surmise that the anti-JEV properties of 25-HC stem from its effects on the host plasma membrane organization and dynamics by modulating membrane cholesterol level, which may impair the entry and fusion of JEV. Given the antiviral effects of 25-HC against Zika and Dengue viruses, our findings suggest that 25-HC could be a promising candidate for developing broad-spectrum anti-flaviviral therapeutics.