Abstract
BackgroundNew therapeutic tools and molecular targets are needed for treatment of Japanese encephalitis virus (JEV) infections. JEV requires an α-1 translational frameshift to synthesize the NS1' protein required for viral neuroinvasiveness. Several flavonoids have been shown to possess antiviral activity in vitro against a wide spectrum of viruses. To date, the antiviral activities of flavonol kaempferol (Kae) and isoflavonoid daidzin (Dai) against JEV have not been described.Methodology/Principal FindingsThe 50% cytotoxic concentration (CC50) and 50% effective concentration (EC50) against JEV were investigated in BHK21 cells by MTS reduction. Activity against viral genomic RNA and proteins was measured by real-time RT-PCR and western blotting. The frameshift site RNA-binding characterization was also determined by electrospray ionization mass spectrometry, isothermal titration calorimetry and autodocking analysis. EC50 values of Kae and Dai were 12.6 and 25.9 µM against JEV in cells pretreated before infection, whereas in cells infected before treatment, EC50 was 21.5 and 40.4 µM, respectively. Kae exhibited more potent activity against JEV and RNA binding in cells following internalization through direct inhibition of viral replication and protein expression, indicating that its antiviral activity was principally due to direct virucidal effects. The JEV frameshift site RNA (fsRNA) was selected as a target for assaying Kae and Dai. ITC of fsRNA revealed an apparent Kb value for Kae that was nine fold stronger than that for Dai. This binding was confirmed and localized to the RNA using ESI-MS and autodock analysis. Kae could form non-covalent complexes with fsRNA more easily than Dai could.Conclusions/SignificanceKae demonstrates more potent antiviral activity against JEV than does Dai. The mode of action of Kae as an anti-JEV agent seems to be related to its ability to inactivate virus by binding with JEV fsRNA.
Highlights
Viral infections are important public health problems worldwide, both in developed and developing countries, due to their morbidity and mortality
Cell culture monolayers infected with different DNA or RNA viruses are the most frequently used in vitro models for determining antiviral activity of natural compounds [17]
Anti-Japanese encephalitis virus (JEV) activity of Kae and Dai in BHK21 cells was analyzed by MTS reduction assay differed markedly from each other in different infection time points, both agents showed dose-dependent anti-JEV activity
Summary
Viral infections are important public health problems worldwide, both in developed and developing countries, due to their morbidity and mortality. Japanese encephalitis virus (JEV) is a leading member of the mosquito-transmitted flavivirus family, and is mainly distributed in China, India and Southeast Asia, where it can cause the central nervous system disease with irreversible neurological damage in humans [1]. There are 30,000–50,000 cases of human Japanese encephalitis worldwide and 10,000– 15,000 deaths each year. JEV is one of the main causes of infectious reproductive failure in swine, resulting in significant economic losses in the pig industry. This virus has a normal zoonotic transmission cycle between swine or birds and mosquitoes. Swine are the main amplifier hosts, from which infected mosquitoes transmit the virus to humans [3,4]. The antiviral activities of flavonol kaempferol (Kae) and isoflavonoid daidzin (Dai) against JEV have not been described
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