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[目的]了解2008年青海省手足口病(HFMD)的主要致病病原体及基因特征和儿童人肠道病毒71型(HEV71)的抗体水平及自然感染状况。[方法]用RT-PCR进行病毒核酸检测,病毒核酸阳性标本进行病毒分离,阳性分离株病毒进行肠道病毒血清型别的分子定型。对分离到的HEV71阳性分离株进行VP1编码区基因扩增,核苷酸序列测定和同源进化分析。对181名1~6岁儿童血清标本,用中和试验检测HEV71中和抗体,并对检测结果进行统计学分析。[结果]517份标本经RT-PCR检测,HEV71核酸阳性50例、CoxA16核酸阳性13例,阳性率为12.2%。分离到45株病毒,其中HEV7130株(占66.7%),是主要病原体。经VP1区核苷酸序列测定后,它们在VP1区核苷酸水平和氨基酸水平上同源性分别在95.2%~100.0%和96.6%~100%之间,与1998年以来在我国分离的HEV71在核苷酸和氨基酸水平上的同源性都较高。与HEV71代表株构建的亲缘进化树中显示,青海省HEV71分离株属于C4基因亚型C4a进化分支。181名1~6岁儿童中HEV71中和抗体阳性率为37.6%,HEV71中和抗体几何平均滴度(GMT)水平为1︰16.12。随着年龄的增大,抗体阳性率及GMT升高,各年龄组抗体阳性率、GMT差异有统计学意义(χ2=41.89,P﹤0.05;r=0.95,P﹤0.05)。[结论]青海省2008年HFMD主要病原为HEV71,其基因型为C4基因型中的C4a进化分支,并且存在多个传播链。1~6岁儿童尤其是≤3岁儿童HEV71中和抗体阳性率低,是HEV71导致HFMD的易感者,是目前手足口病防控的重点人群,加强对HEV71的分子流行病学和血清流行病学监测,对预防和控制HFMD暴发具有重要意义。
[Objective] To understand the main pathogens and genetic characteristics of hand, foot and mouth disease (HFMD) in Qinghai Province in 2008 and the antibody level and natural infection status of human enterovirus 71 (HEV71). [Method] The viral nucleic acid was detected by RT-PCR. Viruses were isolated from positive samples of virus nucleic acid. The positive isolates were serotyped by serotypes of enteroviruses. VP1 coding region gene amplification, nucleotide sequence determination and homologous evolution analysis were performed on the isolated HEV71 positive isolates. 181 serum samples of children aged 1 to 6 were tested for neutralizing antibody against HEV71 by neutralization test and the results were statistically analyzed. [Results] The 517 samples were detected by RT-PCR. Among them, 50 were positive for HEV71 and 13 were positive for CoxA16. The positive rate was 12.2%. 45 strains of viruses were isolated, of which HEV7130 strains (66.7%) were the major pathogens. The nucleotide sequence of VP1 region and its homology in the VP1 region were between 95.2% ~ 100.0% and 96.6% ~ 100%, respectively, which were in line with those of HEV71 isolated in China since 1998 At nucleotide and amino acid levels are high homology. The phylogenetic tree constructed with the HEV71 representative strain showed that the HEV71 isolate in Qinghai belongs to the C4 clade C4a evolutionary branch. The positive rate of HEV71 neutralizing antibody in 181 children aged 1-6 was 37.6%. The geometric mean titer (GMT) of HEV71 neutralizing antibody was 1︰16.12. The antibody positive rate and GMT increased with age. The antibody positive rate and GMT in each age group had significant difference (χ2 = 41.89, P <0.05; r = 0.95, P <0.05). [Conclusion] The main pathogen of HFMD in Qinghai Province in 2008 was HEV71, whose genotype was C4a evolutionary branch in C4 genotype, and there were multiple transmission chains. The positive rate of HEV71 neutralizing antibody in children aged 1 ~ 6 years, especially in children ≤3 years old, is a susceptible to HFMD caused by HEV71 and is the key population for the prevention and control of hand, foot and mouth disease, and enhances the molecular epidemiology and seropositivity of HEV71 Surveillance is important for the prevention and control of HFMD outbreaks.