Influence Of VP1 Protein A289T Mutation On Enterovirus 71 Neurological Infection

Background and ObejectiveEnterovirus 71(EV71)is the main cause of hand,foot and mouth disease,especially the major causes of severe hand,foot and mouth disease(more than 80%).Hand,foot and mouth disease critically ill patients are often associated with EV71 nerve system infection,and easy to leave neurological sequelae,or even death.At present,EV71 has even become the most important central nervous system virus beyond the poliovirus,so the prevention of EV71 neurological infection is the key to reduce the mortality of hand,foot and mouth disease,but also the focus of hand,foot and mouth disease prevention and treatment.Related studies have found that the virulence of the EV71 virus is closely related to the mutation of the amino acids encoded inside the viral genome.The literature proves that VIM is a cell surface EV71 receptor.In this study,the epidemiological study of EV71 viral VP1 A289T is closely related to the occurrence of severe hand,foot and mouth disease.When the amino acid of 289 is Aalanine,the high incidence with the EV71 infects the nervous system(P<0.05,OR =2.36,95%CI 1.163-4.659).It was vertify that VIM is also the receptor of the EV71 on the surface of the HBMEC and affects the replication and release of the EV71 in the cell.This study intends to further study the above results by biological experiments.The virulence and its characteristics of the virus before and after the mutation were studied by reverse genetics,and the effect of A289T mutation of VP1 protein on the nervous system of EV71 virus infection was explored By using the experiment in vitro and animal models,to verify VIM affact EV71 vrus infection.Methods1.EV71 virus site mutation and virus rescueThe EV71-289A infective plasmid was amplified by molecular cloning technique and reverse genetics.Wild-type plasmids and mutant plasmids were transcribed in vitro to obtain RNA,and the viral RNA was transfected into the host cells to obtain two rescue viruses.2.Detection of biological characteristics of virusThe two kinds of viruses was infected with RD cells,and the virus infection,activity,stability and virus quantitative detection were carried out by using cell technology,PCR and Western blot.The infection of the two viruses in HBMEC cells was also compared.3.Virus infected animal modelThe animal model was established and two kinds of rescue viruses were infected by intraperitoneal injection.The pathological changes of the mice were observed and the incidence of the two viruses was counted.The virus was quantitated in the brain tissue of mice by real-time fluorescence quantitative PCR.HE staining Brain tissue lesions;the immunohistochemical method to detect the viral particles in the brain;and the virulence or characteristics of the virus was compared.Result1.EV71-289T infection clone was mutated successfully;the rescue EV71A and EV71T virus was completed.2.There was no significant difference between the two kinds of virus in the invasion,replication and release of RD cells and the virus self-protein synthesis(P>0.05).3.Two kinds of rescue virus could infect HBMEC cells.The EV71A virus in the same MOI infected HBMEC cells was more than the EV71T virus(P<0.05).The adsorption capacity of EV71 virus in control cells was 1.2 times that of vim-ko cells[con:EV71A(1.00±0.15),EV71T(0.58±0.14),vim-ko:EV71A(0.75±0.06),EV71T(0.47 ± 0.20)].MOI=10,virus infect control cells 24h,48h,intracellular EV71A nucleic acid content were 1.3 times,3 times that EV71T nucleic acid,respectively[24h:EV71A(13.7±0.63),EV71T(10.62±0.64),48h:EV71A(100.42± 0.85),EV71T(30.48 ± 0.42)],vim-ko cells were 1.7times,1.8 times[24h:EV71A(2.94 ± 0.96),EV71T(1.72 ± 0.88),48h:EV71A(6.58 ±0.48),EV71T(3.58±0.81)](P<0.05).In cell supernatant,the viral RNA content of EV71A was 2.2 times and 1.6 times higher than that of EV71T,respectively[24h:EV71A(1.00 ± 0.05),EV71T(0.44 ± 0.05),48h:EV71A(8.82 ±0.16),EV71T(5.35±0.07)].The viral RNA content of vim-ko was 1.4 times and 1.2 times higher than that of control cells[24h:EV71A(0.27±0.05),EV71T(0.18 ±0.08),48h:EV71A(0.68±0.06),EV71T(0.54±0.05)](P<0.05).4.The incidence of virus infected BABL/c mice:108pfu virus:the incidence rate of EV71A group was 95.12%(39/41),the incidence rate of EV71T group was 60.47%(26/43);107pfu virus:the incidence rate of EV71A group was 36.36%(8/22)and EV71T group was 11.76%(2/17)(P<0.05).5.The incidence of Virus infected SV129 mice:VIM+/+SV129 mice:108pfu virus:the incidence of EV71A group was 69.23%(27/39),the incidence rate of EV71T group was 42.5%(17/40);10pfu virus:the incidence of EV71A and EV71T were 52.78%(19/36),22.86%(8/35),respectively;VIM-/-129 mice:108 pfu virus,the incidence of both viruses was 10%(1/10);107pfu virus,the incidence of two experimental groups were zero.(P<0.05).6.The virus content in brain tissue of EV71A group was 9.5 times that EV71T group[EV71A(2256.66±50.22),EV71T(237.75±36.15)](P<0.05).Conclusion1.The mutation of VP1 protein 289 amino acid(A to T),the virus virulence was weaken.2.It was verified that the VIM protein was the EV71 receptor of HBMEC cells and the VIM protein was also the receptor of EV71 in SV129 mice.VIM affects EV71 virus adhesion,replication and virus release.