病毒学报

手足口病(Hand-foot-and-mouth disease,HFMD)是5岁以下婴幼儿常见的病毒性肠道传染病,该病主要由人肠道病毒71型(Enterovirus 71,EV71)型及柯萨奇A组16型(Coxsackievirus A16,CV-A16)引起,但关于TLR3、TLR4基因多态性与EV71感染手足口病的报道较少。为探讨EV71感染手足口病患儿TLR3和TLR4基因多态性与EV71感染手足口病严重性及易感性的关系,本研究选择2016年8月至2017年8月就诊于安徽医科大学第一附属医院的EV71感染手足口病患儿166例,其中重症组76例,轻症组90例,并选择同期来院体检的健康者120例作为对照组。收集患者入院时的年龄、性别、发热天数等基线资料,采集血液检测白细胞计数(White blood cell count,WBC)、丙氨酸转氨酶(Alanine aminotransferase,ALT)、谷草转氨酶(Glutamate transaminase,AST)、酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)检测血清C反应蛋白(C reactive protein,CRP)、干扰素-γ(Interferon-γ,IFN-γ)水平;分离外周血单个核细胞提取DNA,琼脂糖凝胶电泳检测DNA情况;聚合酶链反应(Polymerase chain reaction,PCR)扩增TLR3c.1377C/T和TLR4-896A/G,限制性内切酶Tap I(TthHB8 I)、Nco I分别酶切TLR3、TLR4 PCR扩增产物,凝胶成像系统记录实验结果,对扩增产物进行测序,分析其基因多态性结果。结果显示,对照组与EV71感染组TLR3c.1377C/T位点的基因型分布与C、T等位基因频率均无显著统计学意义(P>0.05);EV71感染组中重症组TT基因型较轻症组显著升高(P<0.05);重症组T等位基因频率显著高于轻症组(P<0.05),C等位基因频率显著低于轻症组(P<0.05);EV71感染组中,TLR3c.1377C/T位点不同基因型患儿在年龄、性别及ALT、AST、CKMB水平上无显著差异(P>0.05);TLR3c.1377C/T位点TT型患儿的发热时间及WBC、CRP水平显著高于CT和CC型,CT型患儿的发热时间及WBC、CRP水平显著高于CC型(P<0.05);CC型患儿的IFN-γ水平显著高于CT和TT型(P<0.05);TLR4-896A/G基因电泳条带为140bp的特异性扩增产物,为野生型Asp/Asp基因型,对照组和EV71感染组均未出现A→G的突变。本研究得出结论,TLR3c.1377C/T位点有CC、TT、CT三个基因型,且携带T等位基因EV71感染手足口病患儿进展为重症的风险较高;TLR4-896A/G基因无突变,与EV71感染手足口病患儿疾病严重性和易感性无关。

Hand-foot-and-mouth disease(HFMD)is a common viral enteric infectious disease in children under5 years old. It is mainly caused by human enterovirus 71(EV71)and Coxsackie A16(CV-A16). However,there are few reports about TLR3,TLR4 gene polymorphism and HFMD infected by EV71. Therefore,the relationship between TLR3 and TLR4 gene polymorphism in children with HFMD infected by EV71 and the severity and susceptibility of HFMD were detected and analyzed in this study. A total of 166 children with HFMD infected by EV71 were selected First Affiliated Hospital of Anhui Medical University,China from August 2016 to August 2017:76 in severe group and 90 in mild group. Another 120 healthy people who came to the hospital for physical examination during the same period were selected as the control group. Age,sex,and number of days with fever upon hospital admission were documented. The white blood cell count(WBC)was recorded,as were levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST). Enzyme-linked immunosorbent assays(ELISA)were used to measure serum levels of C-reactive protein(CRP)and interferon(IFN)-γ. DNA was extracted from peripheral blood mononuclear cells,and DNA was detected by agarose gel electrophoresis. TLR3 c.1377 C/T and TLR4-896 A/G were amplified by polymerase chain reaction(PCR). The PCR-amplification products of TLR3 and TLR4 were digested by restriction endonuclease Tap(TthHB8 I)and Nco I,respectively. A gel-imaging system recorded the experimental results. Sequencing of amplified products was done for analyses of gene polymorphisms. Results showed that there was no significant difference in the genotype distribution of the TLR3 c.1377 C/T locus and C、T alleles between the control group and the EV71-infection group(P > 0.05). The frequency of the TT genotype in the severe group was significantly higher than that in the mild group(P < 0.05). The frequency of the T allele in the severe group was significantly higher than that in the mild group,and the frequency of the C allele was significantly lower than that in the mild group(P < 0.05). In the EV71-infection group,there were no significant differences in age,sex or levels of ALT,AST,or CKMB among children with different genotypes of the TLR3 c.1377 C/T locus(P > 0.05). The duration of fever, WBC count and CRP level of TT-type children with a TLR3 c. 1377 C/T locus were significantly higher than those of CT-and CC-type children. The duration of fever,WBC count and CRP levels of CT-type children were significantly higher than those of CC-type children(P < 0.05). The level of IFN-γ in CC-type children was significantly higher than that in CT-and TT-type children(P < 0.05). The TLR4-896 A/G electrophoresis band was a 140 bp-specific amplified product,which had a wild-type Asp/Asp genotype,and there was no A→G mutation in the control group or EV71-infection group. The conclusion of this study is the TLR3 c. 1377 C/T locus has three genotypes:CC,TT,and CT.Children infected with HFMD with a T-allele EV71 infection have a higher risk of developing severe disease.There was no mutation in TLR4-896 A/G,and this was not related to the severity or susceptibility of HFMD in children infected with EV71.