山东省潍坊市柯萨奇病毒A6型VP1区基因进化特征分析

Analysis on characteristics of gene evolution in the VP1 region of Coxsackievirus type A6 in Weifang city,Shandong province

目的 探讨2015-2020年山东省潍坊市柯萨奇病毒A6(CV-A6)VP1区核苷酸进化特征,明确潍坊市CV-A6的基本流行状况及VP1区毒力相关的氨基酸位点。方法 收集2015-2020年山东省潍坊市手足口病患者CV-A6核酸阳性标本,进行VP1区全长序列扩增及核苷酸序列测定,根据GenBank中CV-A6原型株及参考序列(含重症、轻症)进行基因分型、选择性压力分析、重症及轻症间氨基酸位点差异对比分析。结果 共获得15条915 bp VP1轻症全长序列,15条序列均位于D3a分支中,且分别与不同地域序列处于不同分支中。与轻症序列相比,重症序列VP1区的5个氨基酸替代位点S5A(χ^(2)=13.37,P=0.001)、A30V(χ^(2)=6.79,P=0.009)、N137S(χ^(2)=5.40,P=0.02)、V242I(χ^(2)=14.41,P=0.000)、A283T(χ^(2)=14.26,P=0.000)差异均有统计学意义(P<0.05)。潍坊市CV-A6 VP1区序列中,有4条序列在两个位点与重症序列突变一致(I174V、A283T)。所有VP1序列只存在负选择压力,Ka/Ks>1,3个碱基A410G、A520G、G724A发生非同义突变,分别位于三个氨基酸替代位点N137S、I174V、V242I的密码子中,突变率与氨基酸替代率一致。结论 2015-2020年山东省潍坊市手足口病CV-A6 VP1区为轻症序列,均位于D3a分支中D3a基因亚型。重症序列中的N137S、I174V、V242I三个氨基酸位点可能与CV-A6致病性相关。

Objective To explore the phylogenetic features of CV-A6 VPl region sequence in Weifang city from 2015 to 2020,so as to clarify the epidemiological characteristics and virulence related amino acid substitution of Coxsackievirus group A type 6(CV-A6)in Weifang city.Methods Nucleic acid-positive specimens of CV-A6 from patients with hand,foot and mouth disease(HFMD)in Weifang city,Shandong province from 2015 to 2020 were collected for amplification of the full-length sequence of the VPl region and nucleotide sequencing,and genotyping,selective pressure analysis,and comparative analysis of amino acid loci differences between severe and mild cases were performed according to the prototype strain of CV-A6 and reference sequences in GenBank(including severe and mild cases).Results A total of 15 fulllength sequences of 915 bp VP1 mild disease were obtained;All the 15 sequences were located in D3a branch,and they were in different branches with different regional sequences.There were 5 amino acid substitutions of severe sequences:S5A(χ^(2)=13.37,P=0.001),A30V(χ^(2)=6.79,P=0.009),N137S(χ^(2)=5.40,P=0.02),V242I(χ^(2)=14.41,P=0.000),A283T(χ^(2)=14.26,P=0.000)which were compared with mild cases reached statistical significance(P<O.05).Four of the sequences in the CV-A6 VP1 region of Weifang city were consistent with severe sequence mutations at 2 sites(I174V,A283T);All of sequences undergo the strong negative selection pressure,nucleotides of VP1 regeion's Ka/Ks>1,3 Nonsynonymous substitutions(A410G,A520G,G724A)located respectively in codons of 3 amino acid substitution sites(N137S,I174V,V2421),the substitution rates were consistent with amino acid substitution rates.Conclusion From 2015 to 2020,the CV-A6 VP1 region of HFMD in Weifang city,Shandong province is a mild sequence,all located in the D3a subtype of D3a gene in the D3a branch.The 3 amino acid sites N137S,I174V,and V2421 in severe sequence may be related to the pathogenicity of CV-A6.