Inhibition Of Japaneses Encephalitis Virus Infection By Arginine-Rich Peptides Derived From Flaviviridae Helicases

Japanese encephalitis virus (JEV) belongs to Flaviviridae family. It is one of the important pathogens that cause acute viral encephalitis in humans and animals. The JEV is mainly transmitted by the mosquito between animals and human hosts. In humans, the disease can cause irreversible neurologic damage with fatality rates ranging from20%to as high as50%. The disease can cause abortion and stillbirth of sows, orchiditis of boar. The disease severely affected human health and pig industry. The JEV genome encodes3structural proteins and7non-structural proteins, the non-structural protein NS3is a large multifunctional protein possessing protease and NTPase/helicase activities. It plays critical roles in the processing of viral polyprotein and virus genome replication. The intact enzymatic properties of NS3are necessary for the virus propagation, suggesting NS3appears to be a promising target for antiviral agents.The antiviral peptides are a group of small peptides that can inhibit viruse infection. They can inhibit viruse infection in the early stage. More recent works have demonstrated the usefulness of peptides against numerous viral infections, including HIV, hepatitis C virus (HCV), Avian influenza virus (AIV). Antiviral peptides will have broad application prospects. It has been reported that a conserved arginine-rich peptide derived from NS3helicase of HCV, JEV and West Nile virus (WNV) can inhibit the helicase activity.To explore the antiviral activity of arginine-rich peptides from Flaviviride family against JEV infection, we synthesized five peptides corresponding to motif VI on JEV, HCV, WNV, Classical swine fever virus (CSFV) and dengue fever virus (DENV) helicases in this study. We evaluated the inhibitory effects of these peptides toward Japanese encephalitis virus infection by Real-time quantitative reverse transcription-PCR, flow cytometry, plaque assay, animal expermeints, histopathological studies and immunohistochemical detection.Our results showed that these peptides could efficiently penetrate cell membranes quickly and localize in cell ctyoplsm, and the peptides was stable for at least48hours in cells. Theses peptides did not present significant cytotoxicity in BHK-21cells in the concentrations used in our studies. Our experiments showed all of these peptides displayed antiviral activities against Japanese encephalitis virus infection both in vitro and in vivo. The effect concentrations EC50of these peptides were all at micromolar region. Peptides derived from JEV, CSFV and HCV reduced viral RNA level and envelope protein expression at about100μM, inhibited the replication of JEV in BHK-21cells with an EC50at about15μM, while Pdenv and Pwnv have EC50at30and60μM. Animals experiments showed that mice challenged with JEV pretreated with peptides delayed the time of death and reduced clinical symptoms. Moreover, Pjev reduced virus load and mortality in mice. Pjev inhibited JEV infection more effectively than the others, because pretreatment with Pjev provided75%protection against infection. Although other peptides can not protect all the mice from a lethal dose of JEV infection, but delayed the time of death and reduced clinical symptoms.In conclusion, noncytotoxic inhibitory peptides that inhibit JEV infections have been identified. These peptides can effectively inhibit JEV propagation in BHK-21cells. The Pjev could increase mice survival rate when challenged with a lethal dose of JEV when pretreat with the pepide. The peptide Pjev exhibits a high therapeutic index and in vivo efficacy. Pjev, Phcv and Pcsfv were the potent inhibitors against JEV infection. With some modifications, these inhibitory peptides may serve as peptide-based drugs. In the near future, these short peptides could be developed as a potent inhibitor for Japanese encephalitis virus infection and served as the basis for developing novel therapeutics for Japanese encephalitis virus infection.