Effects Of Key Amino Acid Mutations In Enterovirus 71 VP1 Protein On Viral Virulence

Enterovirus group A type 71(EV-A71)is the main cause of hand,foot and mouth disease(HFMD)in infants and young children,it belongs to the micro RNA virus family of enteroviruses.EV-A71 infection is common in children under 5 years of age and is characterised by fever,herpes and rashes on the hands,feet and mouth,and in severe cases,neurological complications and even life-threatening illnesses.However,there is still no specific treatment for this disease.The capsid protein VP1 of EV-A71 virus is the most important neutralizing determinant that can determine the antigenicity of the virus.The gene sequence of the VP1 protein has genetic characteristics that correspond to the serotype of the enterovirus,which is the basis for the classification of enteroviruses according to their serotype.The VP1 protein is a key site for recognition,adsorption and binding of EV-A71 to target cells.Previous studies in our laboratory have shown that the structural protein VP1 of EV-A71 can influence viral replication,but the precise localisation of key amino acid sites on the VP1 protein has not been investigated in more depth.Therefore,by comparing the nucleotide sequences of the VP 1 protein of different virulence phenotypes of EV-A71,screening for suitable virulence candidates,and constructing and rescuing new strains containing a single mutation site,the effect of key amino acid mutations on the VP1 protein on virulence can be investigated and provide a basis for further studies on the role of the VP1 protein in the pathogenesis of EV-A71.Purpose1.To compare the amino acid sequences of the strongly virulent strain SDLY107 and the weakly virulent strain SDJN2015-01 of EV-A71 at the VP1 fragment,to screen for virulence candidate sites,and to construct and rescue mutant strains.2.To investigate the role of key amino acid mutations on the VP1 protein in the pathogenesis of EV-A71 by comparing the mutant strain with the parental strain in terms of cell damaging power,replication capacity,cell autophagy,apoptosis and cell infection efficiency.Methods1.The recombinant plasmid containing the mutant site was obtained using the plasmid pMD19T-SDLY107-EGFP as template,overlapping fusion PCR and reverse genetics techniques,and then transfected into BSR-T7/5 cells to obtain the mutant strain after successful rescue.The mutant strain was identified by plasmid double enzyme digestion and sequencing verification,and the virus titer was determined by plaque assay and CCID50.2.Differences in replication capacity,cell damage-causing capacity,cell autophagy,apoptosis and cell infection efficiency between mutant and parental strains in RD and SH-SY5Y cells were examined.Results1.Mutant strains SDLY-EGFP-107(VP1-1)and SDLY-EGFP-107(VP1-2)were successfully constructed and rescued,and their viral titres were measured.2.RT-qPCR results showed that the mutant strains SDLY-EGFP-107(VP1-1)and SDLY-EGFP-107(VP1-2)were less capable of replicating in RD cells and SH-SY5Y cells than the strong strain SDLY107.3.CCK-8 and LDH assays detected that the mutant strains SDLY-EGFP-107(VP1-1)and SDLY-EGFP-107(VP1-2)were significantly less cytotoxic than the strong strain SDLY107 in both RD and SH-SY5Y cells.The mutant strain SDLY-EGFP-107(VP1-2)was less cytotoxic than SDLY-EGFP-107(VP1-1)in RD cells.4.Western Blot results showed that both mutant strains SDLY-EGFP-107(VP1-1)and SDLY-EGFP-107(VP1-2)were able to induce autophagy in RD cells,but failed to significantly affect the level of autophagy in SH-SY5Y cells.5.The results of flow cytometry assay showed that the mutant strains SDLY-EGFP-107(VP1-1)and SDLY-EGFP-107(VP1-2)induced weaker levels of apoptosis and cell infection efficiency than the strong virulent strain SDLY107,and the mutant strain SDLY-EGFP-107(VP1-2)had a weaker effect on both than SDLY-EGFP-107(VP1-1).Conclusion1.Successful construction and rescue of mutant strains SDLY-EGFP-107(VP1-1)and SDLY-EGFP-107(VP1-2).2.Mutations in amino acids 146th and 147th of VP1 protein both attenuated the cytotoxic and replicative power caused by EV-A71 infection and affected the cellular autophagy process of EV-A71 in neuronal cells,and both down-regulated the level of apoptosis after viral infection and reduced the cellular infection efficiency of EV-A71.3.The effect of amino acid mutation at position 147th of VP1 protein on virulence was significantly stronger than that of amino acid mutation at position 146th.