柯萨奇病毒A组6型对利巴韦林耐药性的初步研究

Preliminary Study on Resistance of Coxsackievirus A6 to Ribavirin

柯萨奇病毒A组6型(Coxsackievirus A6,CVA6)已成为近年来国内导致手足口病的主要病原体。本研究挑选16株中国流行的不同基因亚型的CVA6,通过在RD细胞上的细胞活性实验和观察致细胞病变效应来检测利巴韦林对不同CVA6毒株的抑制率。结果表明药物浓度在0.8mmol/L和0.4mmol/L作用下对部分CVA6有明显的抑制作用(P<0.0001),在0.8mmol/L药物浓度下根据抑制率的高低,将抑制率大于80%的CVA6株(5株,占31.25%)以及低于10%的CVA6株(5株,占31.25%)分别定义为对利巴韦林敏感组和耐药组。为了探究CVA6耐药性与病毒致病力之间的关系,在利巴韦林敏感组与利巴韦林耐药组分别随机挑选两株CVA6进行ICR乳鼠感染实验。通过观察乳鼠生存率、临床评分和体重变化,结果显示利巴韦林耐药组CVA6致病力均比利巴韦林敏感组弱,表明利巴韦林耐药与致病力之间存在一定联系。最后使用MEGA软件对利巴韦林敏感组和耐药组CVA6进行3D区氨基酸序列比对分析,发现两组CVA6在3D区75位、308位、342位、346位氨基酸完全不同,提示利巴韦林耐药与3D区的基因多态性存在一定联系。本研究提示对利巴韦林耐药的CVA6广泛存在,属于目前优势流行D3a基因亚型,不仅为手足口病患者个体化治疗提供了重要依据,而且对CVA6感染相关疾病的防控具有重要意义。

In recent years, Coxsackievirus A6(CVA6) has become the main pathogen leading to hand, foot, and mouth disease(HFMD) in China. In this study, 16 CVA6 strains of different sub-genotypes circulating in China were selected to measure percent inhibition of different CVA6 strains by ribavirin using viability experiments on RD cells and observing the cytopathic effect. We discovered that some CVA6 strains were inhibited significantly at ribavirin concentrations of 0.8 mmol/L and 0.4 mmol/L(P<0.0001). At a ribavirin concentration of 0.8 mmol/L, CVA6 strains that were inhibited by >80%(five strains, 31.25%) and CVA6strains inhibited by <10%(five strains, 31.25%) were defined as the “ribavirin-sensitive group” and “ribavirinresistant group”, respectively. To investigate the relationship between CVA6 resistance and viral pathogenicity, two strains of CVA6 were selected randomly from the ribavirin-sensitive group and ribavirin-resistant group for infection into ICR mice. Observation of the survival rate, clinical score and bodyweight change of mice revealed that the pathogenicity of CVA6 in the ribavirin-resistant group was weaker than that in the ribavirin-sensitive group, which indicated a relationship between ribavirin resistance and virulence. Finally, MEGA software was used to compare the amino-acid sequences of CVA6 in 3D region between the ribavirin-sensitive group and ribavirin-resistant group. We found that CVA6 in the 3D region was different at amino acids 75, 308, 342 and 346 in the two groups, which suggested that resistance to ribavirin was associated with genetic mutations in this 3D region. Our study suggested that ribavirin-resistant CVA6 was widespread and belonged to the dominant epidemic D3a sub-genotype at present. which not only provides an important basis for individualized treatment of HFMD patients, but also has important significance for prevention and control of CVA6 infection-related diseases.