Preliminary Study on the Effect of Coxsackievirus A16 VP1-145 Amino Acid Mutation on Heparan Sulfate Binding Ability
Coxsackievirus A16(CVA16)and enterovirus A71(EV-A71)are important pathogens causing hand,foot and mouth disease(HFMD)in China.They are homologous and from a common ancestor,with the same genome structure,and both use heparan sulfate(HS)as an adsorption receptor.Based on the research regarding of VP1-145 amino acid site of EV-A71,three strains of CVA16 with differences in VP1-145 amino acid site selected in this study that were prevalent in China to study the impact of VP 1-145 amino acid site variation on HS binding ability.Human rhabdomyosarcoma(RD)cells were treated with different concentrations of heparinase Ⅰ,and the adsorption ability of different CVA16 strains on RD cells before and after heparinase Ⅰ treatment was compared,as well as the replication of each strain on RD cells 12h,24h and 48h after inoculation.The results showed that under the treatment of heparinase Ⅰ at concentrations of 5 mIU/mL and 10 mIU/mL,the adsorption ability of CVA16 with the amino acid Glycine(G)at VP1-145 site to RD cells decreased,and the virus titer decreased at 12h,24h and 48h after inoculation.The degree of decrease was positively correlated with the concentration of heparinase Ⅰ,and the difference was statistically significant(P＜0.05).However,no such trend was observed in the two CVA16 strains with glutamic acid(E)at VP1-145 site.In order to further explore the relationship between the differences in binding ability and the virulence of the viruses,two-day-old ICR suckling mice infection experiment was conducted using three virus strains.To evaluate the differences in pathogenicity of different CVA16 strains in mouse infection models,the survival rate,clinical scores,and weight change of suckling mice were observed.The results showed that the pathogenicity of CVA16(VP1-145E)was significantly stronger than that of CVA16(VP1-145G).Then the whole genome sequencing of the three CVA16 strains showed that there were other different sites in addition to the VP1-145 site.Therefore,we rescued the virus by reverse genetics and further explored the mechanisms of the VP1-145 site.The results of this study confirmed that the amino acid variation of VP1-145 site could affect the binding ability of CVA16 to HS,but whether it is the cause of the difference in the pathogenicity of CVA16 in suckling mice needs to be further explored by animal experiments.This study provides a theoretical basis for the study of the virulence sites and pathogenic mechanisms of CVA16.
NETL
NSTL
万方数据
