Effects Of Key Amino Acid Mutations Of Enterovirus 71 3D Protein On Virus Replication

Hand,foot and mouth disease(HFMD)is caused by enteroviruses,especially Enterovirus A group 71(EV-A71)and Coxsackievirus A group 16(CV-A16)are the most common.Infection with EV-A71 can have two different clinical symptoms,mild and severe.Mild symptoms include fever,sore throat,and skin rash.Most are self-limiting and can heal itself within a few days.Severe symptoms include aseptic meningitis,acute flaccid paralysis,cardiopulmonary complications,and neonatal sepsis.It can be accompanied by central nervous system complications and even death.It is easy to misjudge whether the rescue is successful through the cytopathic effect(CPE).Therefore,The laboratory constructed a recombinant virus labeled with green fluorescent protein(EGFP),and the replication kinetics test showed that the recombinant virus did not affect the biological activity of the virus.In the previous study,it was found that after replacing the 3D coding region of the attenuated strain SDLY1 into the virulent strain SDLY107,the replication ability of the replacement strain was weaker than that of the virulent strain.Therefore,it is speculated that there are sites in the 3D coding region that affect the ability of the virus to replicate,and 5 different amino acid sites of the strong and weak virus 3D proteins have been screened out.At the same time,it was found in the apoptosis rate test that the 3D protein transfection group of SDLY107 strain had a higher apoptosis rate than the negative control group.There are sites in the 3D protein that affect cell apoptosis.This study used homologous recombination site-directed mutagenesis technology and reverse genetics technology to construct and rescue mutant strains EGFP-EV-A71(3D-1)and EGFP-EV-A71(3D-2).Compare its ability to replicate,cause cell damage,and induce cell apoptosis.At the same time,pymol software is used to draw 3D protein structure model diagrams,to more intuitively understand the protein structure changes after site mutations,the effects and the effect of protein structure changes on virus replication.It provides reference for the research on the pathogenic mechanism of EV-A71 virus,the research and development of antiviral drugs and the preparation of vaccines.Objective:1.To screen the 3D protein virulence candidate sites of EV-A71,construct and rescue the mutant strain EGFP-EV-A71(3D-N).2.To compare the replication kinetics characteristics of the virus strain before and after mutation and its effect on cell apoptosis,and explore the role of 3D protein virulence candidate sites in the pathogenic mechanism of EV-A71.Methods:1.The difference in growth kinetics between SDLY107 strain and recombinant strain was determined by qRT-PCR,LDH and CCK-8 methods.2.PCR,homologous recombination,site-directed mutagenesis,and reverse genetics are used to obtain target fragments,mutant plasmids,and mutant strain viruses.The mutant viruses are identified by double enzyme digestion of the mutant plasmid,green fluorescent protein expression and sequencing verification.CCID50 is used to determine virus titer.3.By detecting the replication kinetics indicators of different strains in different cells,flow cytometry is used to determine the rate of apoptosis,compare the differences between the strains,and determine the changes in virus replication ability and apoptosis rate of mutant strains.4.The pymol software is used to draw the 3D protein Surface mosaic model and the Cartoon model,and analyze the changes in the 3D protein structure after site mutation and the effect of the structural changes on the virus replication ability.Results:1.The recombinant strain EGFP-EV-A71 with green fluorescent protein marker is similar to the parent strain SDLY107 in terms of replication and cell damage,and can replace the parent strain for further research.2.The mutant strains EGFP-EV-A71(3D-1)and EGFP-EV-A71(3D-2)were constructed and successfully rescued.Back mutation verification successfully constructed a cDNA clone containing mutations at E245G and G354A sites.3.The results of the replication curve determined by the qRT-PCR method showed that:in RD cells,no significant difference was detected in the replication ability of each strain;in Vero cells,the replication ability of the mutant strain was weakened;in SH-SY5Y cells,mutation strain was significantly weakened,and the viral load was always at a low level during the detection cycle.4.The results determined by LDH and CCK-8 showed that the cytotoxicity of each strain is the same in the three kinds of cells;the mutant strains EGFP-EV-A71(3D-1)and EGFP-EV-A71(3D-2))is the weakest,and both are weaker than the attenuated SDLY1.And the difference was not statistically significant(P<0.05).5.Apoptosis detection results showed that in RD cells,SDLY1,SDLY107 and EGFP-EV-A71 can cause higher levels of apoptosis after infection.The mutant strain EGFP-EV-A71(3D-1)and EGFP caused a decrease in the level of apoptosis after infection.In Vero cells,the mutant strain EGFP-EV-A71(3D-1)and EGFP-EV-A71(3D-2)caused a lower level of apoptosis after infection than EGFP-EV-A71 and SDLY107.Conclusions:1.EV-A71 3D protein S21N,R126K mutations have an impact on the replication of the virus in cells,especially in nerve cells,the replication level of the mutant virus is significantly reduced.2.The 3D protein E245G and G354A mutations cause the virus to be unable to rescue,which may be a lethal mutation of EV-A71.