Screening of Natural Extracts for Inhibitors against Japanese Encephalitis Virus Infection

Guo, Jiao; Jia, Xiaoying; Liu, Yang; Wang, Shaobo; Cao, Junyuan; Zhang, Bo; Xiao, Gengfu; Wang, Wei

Ouabain protects against JEV infection-induced lethality in vivo. As ouabain exhibited a stronger inhibitory activity on JEV infections, we further examined the protective effect of ouabain on JEV-induced lethality by using the BALB/c mouse. As anticipated, mice in the JEV-infected, vehicle-treated group started to show symptoms To further relate these protective effects to viral load and histopathological changes in mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at days 7, 10, and 21 postinfection. The results indicated that, during the progression of the disease, ouabain treatment significantly reduced viral load in infected mice compared to untreated infected mice. Notably, no plaques formed in the ouabain-treated group on day 21 postinfection (Fig. 6B) . Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, and vacuolar degeneration, was observed in the JEV-infected and vehicle-treated group on day 7 postinfection, while ouabain treatment remarkably alleviated these phenomena (Fig. 6C) . These results indicate that alleviation of histopathological changes is accompanied by a reduction in the viral load as well as a reduction in mortality rate, further confirming the curative effects of ouabain on viral encephalitis. The viral loads of brain, blood, and spleen were also evaluated at the early stage of infection. As shown in Fig. 6D , on day 1 postinfection, viral titers in the brain were almost undetectable, and ouabain exhibited little effect on viral titers in the brain at the preclinical stage. Accordingly, ouabain had little effect on decreasing viral titers in the blood and spleen at the earlier time points, which was mostly due to that the detectable viral genome copy numbers were too low to be comparable. Notably, titers in the blood decreased sharply from day 1 to day 3 postinfection, which was in line with the characteristic viremia caused by JEV. These results indicate that ouabain could alleviate histopathological changes and reduce viral loads in the brain, thus protecting mice against JEV-induced lethality and confirming the curative effects of ouabain on viral encephalitis. FGIN-1-27, an anxiolytic drug that targets the peripheral benzodiazepine receptor, reduced the JEV infection in vitro (15) . Drug screening and repurposing has become a very useful approach for identifying antiviral drugs, as it explores novel molecular targets to study virus pathogenesis.