The Study Of Inhibition Of Japanese Encephalitis Virus Infection By NSK Tripeptide

Japanese encephalitis (JE) caused by Japanese encephalitis virus (JEV),is usually infected to the human body and animal through mosquitoes and then may cause the encephalitis and neurologic disease. JEV belongs to the genus Flavivirus which also include West Nile virus and Dengue virus. Swine is considered the main reservoir and amplifier of Japanese encephalitis virus in the natural environment. Japanese encephalitis is one of the main diseases of Swine, which can cause abortion and stillbirth of sows, orchiditis of boar. Hence,it is essential to prevent swine JE,which is useful for swine production as well as health of human being. With the biology developing,antiviral peptides are a kind of small peptide molecules with biological activity which can destsroy or inhibit virus particals.Because of their molecular structure and mechanism of action,antivirial peptides have been widely studied. In vitro or in vivo experiments have shown that some peptides have good antiviral effect.Viral Replication cycle is divided into five parts, adsorption, penetration, uncoating,synthesis of macromolecule and assembly and release. Any parts are blocked,then the virus paritcal cannot be produced,and the drugs will be an antiviral drugs. E protein is the envelope glycosylated protein and the major structural protein on the surface of the mature JE virions. E protein is essential for membrane fusion and mediates binding to host cells. One of mechanism of antiviral drugs is that interact with host cell receptors to block virus entry. The domain III of viral envelope protein (EDIII) plays important roles in interacting with host cell receptors to facilitate virus attachment.Hence the EDIII protein and loop3peptide exert inhibitory effects on virus infection by interfering in virus attachment to the host cells.The studies presented here showed that the NSK tripeptide, which is a part of domain III of viral envelope protein (EDIII) and loop3peptide exert inhibitory effects on virus infection by interfering in virus attachment to the host cells. The contents of the paper contain five parts as following:1. Base on the plasmid pCold-NsuA-EDIII loop3and using the method of quick-change,we mutated the363-369non-alanine in loop3to alanine and constructed the recominbinant plasmid pCold-NsuA-EDIII-T363A, pCold-NsuA-EDIII-S364A, pCold-NsuA-EDIII-S365A, pCold-NsuA-EDIII-N367A, pCold-NsuA-EDIII-S368A, pCold-NsuA-EDIII-K369A.The the recominbinant Prokaryotic expression plasmid was transformed into E.coli Rosetta2, then they were expressed. Western blot showed all the expressed proteins have anti-JEV activity, which laid foundation for the subsequent tests.2.Selecting the JEV which were proliferated by cell culture of BHK-21as the study target, using Pierce*BCA*Protein Assay to observe CPE,determine the D490nm and TCID50, plaque formation assay to decided the inhibition rate to JEV by the different EDIII loop3protein.The results showed that the inhibition rate to JEV by EDIII-N367A,EDIII-S368A, EDIII-K369A significantly decreased.3.Infected with JEV after the BHK-21cells were treated with different concentrations of NSK peptide or KNS peptide,we measured the antiviral acticities of NSK peptide determined by plaque formation assay,Q-PCR and Western blot analysis at48hpi.The result showed that NSK peptide had the significant inhibitory effect against JEV infection,especially the concentration is100p.g/ml,also in a dose dependent manner.Certainly,the result suggested that the inhibitory ability of the NSK peptide is sequence-specific.4. As the NSK peptide was not cytotoxic, next we determined whether the NSK peptide could protect mice from lethal dose challenge of JEV.BALB/c mice were injected intraperitoneally or intracerebrally.The group of injected intracerebrally survival data suggested that the NSK peptide can protece mice from death with the survival rate of90%at forth day and of70%from seventh day, and the survival rate still of90%at21th day.The study suggested that the tripeptide NSK derived from loop3of JEV EDIII can inhibit JEV infection.