2014—2022年淄博市手足口病病原谱变化及基因特征分析

Changes in pathogen spectrum and genetic characteristics of HFMD in Zibo city from 2014 to 2022

目的分析山东省淄博市2014—2022年手足口病(hand,foot,and mouth disease,HFMD)病原谱的构成变化,掌握淄博市手足口病主要致病原的流行规律,为手足口病预防控制工作提出建议及参考依据。方法采用实时荧光反转录PCR(RT-PCR)方法对来自2014—2022年间淄博市的4053份咽拭子样本进行肠道病毒核酸检测,选择人横纹肌肉瘤(human rhabdomyosarcoma,RD)细胞培养阳性分离物进行肠道病毒VP1区扩增及测序,从GenBank中选取不同基因型别参考序列,用MAGA 11.0软件中邻接法(neighbor-joining,NJ)构建系统进化树,并采用DNAStar Mega Align软件对VP1区序列进行同源性比对分析。结果共采集淄博市手足口病患儿咽拭子样本4053份,核酸检测结果显示:样本的总阳性率为70.91%,3~6岁的幼托儿童阳性率最高(72.50%);2014年的优势血清型为EV-A71,2015年、2017年和2018年的优势血清型为非EV-A71和CVA16的其他肠道病毒,2016年和2019年的优势血清型为CVA16,2020年之后的优势血清型均为非EV-A71和CVA16的其他肠道病毒;淄博市手足口病发病高峰以5~8月为主,6月为高峰。不同区县的HFMD病原谱分布差异有统计学意义(χ^(2)=48.841,P<0.05);对获得的63份阳性毒株分离物进行测序后比对分析,显示淄博市存在6种肠道病毒CVA10(28.57%)、CVA16(17.46%)、CVA6(7.94%)、CV-A2(6.35%)、CV-A4(12.70%)和EV-A71(26.98%)。进化分析及同源性分析结果显示淄博市HFMD病例EV-A71毒株呈现高度集中,均为C4a亚型,其毒株间VP1区核苷酸同源性为92.50%~100.00%,HFMD病例CVA16毒株均属于B1亚型,其中B1a占36.36%,B1b占63.64%,存在B1a和B1b亚型共流行,其毒株间VP1区核苷酸同源性为88.10%~100.00%。结论2014—2022年淄博市手足口病原谱呈现多样性,2014—2019年,淄博市手足口病优势毒株交替出现,自2020年开始逐渐转变为以非EV-A71非CVA16的其他肠道病毒为主。3~6岁托幼儿童为重点人群,应重点开展其他肠道病毒的检测和VP1区的分型工作,以期了解本地HFMD流行规律,控制疾病流行。

Objective To analyze the changes in the composition of the pathogens for hand,foot,and mouth disease(HFMD)in Zibo city,Shandong province from 2014 to 2022,and aware the epidemic patterns of the main pathogens of HFMD in this city,so as to provide suggestions and reference basis for the prevention and control of HFMD.Methods Real time fluorescent reverse transcription PCR(RT-PCR)was used to detect enterovirus nucleic acid in 4053 pharyngeal swab samples from Zibo city from 2014 to 2022.Positive culture by human rhabdomyosarcoma(RD)cell culture was selected for amplification and sequencing of the enterovirus VP1 region.Reference sequences of different genotypes were selected from Genbank,and a phylogenetic tree by neighbor joining(NJ)method was constructed using MAGA 11.0 software.MegaAlign in DNAStar software was used for VP1 sequence alignment and similarity analysis.Results A total of 4053 throat swab samples were collected from children with HFMD in Zibo city.The nucleic acid test results showed that the total positive rate of the samples was 70.91%,with the highest positive rate detected in children aged 3-6 years old in daycare(72.50%).The dominant serotype in 2014 was EV-A71,while the dominant serotypes in 2015,2017,and 2018 were non-EV-A71 non-CVA16 enteroviruses.CVA16 was the dominant serotypes in 2016 and 2019.In the years after 2020,all dominant serotypes were non EV-A71 non-CVA16 enteroviruses.The incidence peaks of HFMD in the city occurred mainly from May to August with June as the peak.The differences in pathogen spectrum of HFMD in districts and counties were statistically significant(χ^(2)=48.841,P<0.05).The sequencing and comparative analysis of 63 positive isolates revealed that there were six types of enteroviruses in this city,i.e.CVA10(28.57%),CVA16(17.46%),CVA6(7.94%),CV-A2(6.35%),CV-A4(12.70%)and EV-A71(26.98%).The evolutionary analysis and similarity analysis showed that the EV-A71 strains of HFMD cases in Zibo city were highly concentrated and all belonged to C4a subgenotype.The nucleotide similarities of VP1 region among the strains ranged 92.50%-100.00%.All CVA16 strains of HFMD cases belonged to B1 subgenotype.B1a and B1b accounted for 36.36%and 63.64%,respectively.There was co-circulation of B1a and B1b and the nucleotide similarities in the VP1 region among the strains were 88.10%-100.00%.Conclusions The pathogen spectrum of HFMD in Zibo city showed diversity from 2014 to 2022.From 2014 to 2019,the dominant strains of HFMD in Zibo city appeared alternately,gradually changed to non-EV-A71 non-CVA16 enteroviruses after 2020.Children aged 3-6 years old in childcare facilities was the key population.Testing of other enterovirus and VP1 typing should be carried out on focus to understand the local prevalence of HFMD and control the disease.