The Antiviral Research Of Enterovirus 71 And Ebola Virus

Enterovirus 71 belonging to the Picornaviridae family,are non-enveloped single-stranded positive-sense RNA viruses,which is the major causative agents for hand,foot,and mouth disease especially for the neurological complications and fatalities in children.Unfortunately,there is currently no specific anti-EV-A71 drug but limited to supported care.Although two inactivated EV-A71 vaccines have been approved by the China Food and Drug Administration(CFDA),they have yet not to be widely implemented.The pathogenic mechanism of EV-A71 and the important function of VP4 remain unknown.A reverse genetics system of EV-A71 will give some hope to the research of EV-A71 virology and pathogenesis,the system with site-directed mutagenesis on VP4,may contribute to the anti-viral study.To gain insight into the role of positively charged residues in VP4 function during these processes,a charged-to-alanine scaning analysis was performed using an infectious cDNA clone of EV-A71.Seven mutants containing single charged-to-alanine changes were tested.One of them was inviable,four mutants were replication defective,and the remaining two mutants were replication competent.By selecting revertants,second-site mutations which could partially or completely restore viral infectivity were identified on VP1 for two defective mutations,while a second-site mutations within VP4 for one lethal mutations.The results revealed a network of intra-and intermolecular interactions of the VP4 and VP1 protein,which have not been found.Interestingly,we identified a chemical called formonorntin exerting inhibition on EV-A71 by interfering virus uncoating,and a mutation K58 T on VP4 conferred resistance to formonorntin.Except for K52 A,the other VP4 mutants we constructed by charged-to-alanine scaning analysis are less sensitive to formonorntin,suggesting that VP4 mutantsshow some resistance to the chemical targeted on VP4 K58.The VP4 mutants showed lower thermal stability at 42°C compared to wild-type virus except for K69 A.All in all,the five out of seven positively charged residues in VP4 are essential to produce infectious particals.Ebola virus(EBOV)is a highly pathogenic negative strand RNA virus belonging to filoviridae,which caused a Ebola epidemic in Africa arousing a worldwide panic.The treatment of Ebola virus disease is limited to support care because of no approved medicine.We developed a Ebola virus mini-genome system based on EBOV RNP complex to screening a natural product library including 502 chemicals,and found that 9’-cis-retinoic acid is a effective Ebola pseudovirus replication inhibitor.To validate the hits,we introduced a dual-luciferase reporter assay system to exclude cell toxicity and transfection effectivity,the replication of this Ebola pseudovirus still was inbibited by 9’-cis retinoic acid.Otherwise,9’-cis-retinoic acid can inhibit the replication of Respiratory Syncytial Virus(RSV),a negative strand virus similar to Ebola virus both in live virus and RSV mini-genome system.We also tested two9’-cis-retinoic acid analog(13’-cis-retinoic acid and all trans-retinoic acid)that they all exhibit luciferase reduction on Ebola virus mini-genome system.Therefore,retinoic acid compounds have a remarkable prospect in the development of anti-Ebola virus drugs.