Anti- Japanese-Encephalitis-Viral Effects of Kaempferol and Daidzin and Their RNA-Binding Characteristics

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.

Antiviral Activity of Baicalein and Quercetin against the Japanese Encephalitis Virus

“…Indeed, it was demonstrated that baicalein increased the in vitro and in vivo antiviral activity of ribavirin against influenza virus significantly [24]. Recently, through theantiviral activity of kaempferol and daidzin, two types of flavonoids against Japanese encephalitis virus were reported [11]. In the present study we also have demonstrated that baicalein exhibited potent in vitro anti-JEV effects at all different stages of JEV infection compared to the quercetin.…”

“…There have been efforts to find effective antiviral substance among the natural compounds such as plant or algal derived compounds [9,10]. Recently, in vitro anti-JEV activities of two different types of bioflavonoids namely kaempferol and daidzin were evaluated and it was shown that kaempferol is more effective against JEV replication compared to daidzin [11]. Compounds from natural resources are regarded as possible alternatives as they may show low side effects and easily accessible from nature.…”

Inhibition of Enterovirus 71 Replication by 7-Hydroxyflavone and Diisopropyl-Flavon7-yl Phosphate

“…Quercetin, a member of the flavonol subgroup of flavonoids, is capable of inhibiting influenza virus, dengue virus, and JEV [36], [37], [38]. The antiviral activities of kaempferol and daidzin against JEV were also reported by our lab [39]. But to the best of our knowledge, no study has investigated the antiviral properties of HF and FIP from the flavone subgroup in vivo or in vitro.…”

Potential chemotherapeutic targets for Japanese encephalitis: current status of antiviral drug development and future challenges

“…[94,95] Minocycline Antioxidant [99,100] Arctigenin Antioxidant [101] Fenofibrate Antioxidant [102] Curcumin Antioxidant [103] Pentoxifylline Assembly or Release [105] Nitazoxanide Early/mid-replication cycle [107] Nucleic acid-based siRNA C, M, E, NS1, NS3, NS4B, NS5 [109][110][111][112][113][114][115] PNA UTR [119] Morpholino oligomer UTR [121,122] Virus replication cycle-based Heparan sulfate Receptor binding [123][124][125] E-binding peptide Receptor binding [126] NSQ Attachment [127] Indirubin Attachment [128] Bovine lactoferrin Receptor binding [129] Griffithsin Receptor binding [130] Recombinant E Receptor binding [131,132] MCPIP1 RNA replication [133] Kaempferol RNA replication [134] SCH16 Translation [138,139] In silico modeling-based Ivermectin NS3 [146] 4-hydroxypanduratin NS2B/NS3 [147] C randomized, double-blind, human clinical trial conducted in India [95]. A variety of virus-infected cells have been shown to produce reactive oxygen species (ROS), which are associated with viral pathogenicity, for example, by inducing cellular apoptosis [96].…”

“…Monocyte chemoattractant protein 1-induced protein 1 (MCPIP1), which contains a nuclease domain, displays anti-JE activity in vitro, and the RNase, RNA-binding and oligomerization properties of MCPIP1 are required for its anti-JE activity [133]. Kaempferol, a polyhydric flavonol derived from various plants, inhibits JEV replication by binding to the viral genome in vitro [134]. FGIN-1-27, cilnidipine, niclosamide and bispidine--amino acid conjugates displayed anti-JE activities in vitro, probably by inhibiting RNA replication [135,136].…”