柯萨奇病毒A组16型VP1-145位氨基酸变异影响对硫酸乙酰肝素结合能力的初步探究

Preliminary Study on the Effect of Coxsackievirus A16 VP1-145 Amino Acid Mutation on Heparan Sulfate Binding Ability

柯萨奇病毒A组16型(Coxsackievirus A16,CVA16)与肠道病毒A71型(Enterovirus A71,EV-A71)是国内导致手足口病的重要病原体,二者同源,来自共同祖先,基因组结构相同,并且均使用硫酸乙酰肝素(Heparan sulfate,HS)作为吸附受体。本研究基于EV-A71的VP1-145位氨基酸位点的相关研究结果,分析CVA16的VP1-145氨基酸差异,选取3株中国流行的,在该位点具有差异的CVA16,进行对HS结合能力影响的研究。使用不同浓度肝素酶Ⅰ处理人横纹肌肉瘤(Human rhabdomyosarcoma,RD)细胞,比较不同CVA16株对肝素酶Ⅰ处理前后的RD细胞的吸附能力,以及接种后12h,24h,48h后各毒株在RD细胞的复制情况。结果表明,在使用5mIU/mL和10mIU/mL浓度肝素酶Ⅰ水解RD细胞表面的HS后,VP1-145氨基酸位点为甘氨酸(Glycine,G)的CVA16对RD细胞的吸附能力下降,其下降程度与肝素酶Ⅰ的作用浓度呈正相关,且影响了后续12h,24h,48h病毒在RD细胞中的复制,其差异具有统计学意义(P<0.05)。而VP1-145氨基酸位点为谷氨酸(Glutamic acid,E)的两株CVA16均未观察到此种趋势。为了进一步探究该种结合能力的差异与病毒致病力之间的关系,对三株病毒进行2日龄ICR乳鼠感染实验。通过观察乳鼠生存率、临床评分、体重变化,来评估不同CVA16株在小鼠感染模型中的致病力差异。结果表明,CVA16(VP1-145E)的致病力明显强于CVA16(VP1-145G)。后对三株病毒进行全基因组测序,测序结果显示,除VP1-145位点外,三株CVA16还存在其他的差异位点,故后续我们又通过反向遗传学拯救病毒,对VP1-145位点的作用机制进行了进一步的探讨。本研究结果证实,VP1-145位点的氨基酸变异会影响CVA16与HS的结合能力,145位点为E的病毒,其与HS的结合能力较差,当145位点由E突变至G时,病毒与HS的结合能力增强,其与RD细胞的结合能力受到肝素酶Ⅰ的影响。本研究为CVA16毒力位点与致病机制的研究提供了一定的理论基础。

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 VP1-145 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.