一起急性出血性结膜炎疫情病原学鉴定及基因特征分析

Pathogenetic identification and genetic characteristics of an acute hemorrhagic conjunctivitis outbreak

目的 为鉴定2023年深圳市宝安区某学校一起急性出血性结膜炎(acute hemorrhagic conjunctivitis, AHC)聚集性疫情的病原并进行病原学特点分析,为快速追溯传染源以及处置此类疫情提供了参考和依据。方法 对深圳市宝安区2023年某学校一起AHC聚集性疫情采集22份眼拭子标本进行分析。采用实时荧光定量聚合酶链式反应(polymerase chain reaction, PCR)方法对采集自患者的眼拭子标本进行肠道病毒70型(Enterovirus 70, EV70)和柯萨奇病毒A组24型(Coxsackievirus A24, CVA24)基因检测,然后测定阳性样本中病毒基因(VP1)核苷酸的全序列,测定的序列与不同时期、不同地区CVA24的序列比对,分析同源性并构建系统进化树,同时将核苷酸序列翻译成氨基酸序列,分析氨基酸位点突变情况。结果 采集的22份眼拭子标本中,经荧光定量PCR检测,有16份CVA24病毒阳性,阳性率为72.73%,而EV70病毒均为阴性。其中16份标本用VP1引物进行扩增,并对扩增产物测序,成功扩增并测序出14份标本的VP1全长序列。分析和比对核苷酸序列,鉴定结果均为CVA24v GⅣ基因型。基因进化分析显示这14株CVA24v核苷酸同源性为100.00%,与CVA24v的原型株EH24/70的核苷酸同源性为85.14%,与CVA24的原型株Joseph的核苷酸同源性为77.27%。系统进化树显示,它们与同时期广东省中山市流行的CVA24v毒株处于同一进化分支上,其亲缘关系最近。与2023年以前流行的参考序列的氨基酸序列比较,本次流行株的VP1区氨基酸序列在16位点形成一个新的突变,由亮氨酸(L)替换为异亮氨酸(I)。结论 本次急性出血性结膜炎聚集性疫情的病原体是柯萨奇A24型变异株,其氨基酸序列出现一个新的变异位点,根据系统进化树结果,推测毒株可能来源于同时期广东省流行的株型。

Objective To identify the pathogenetic characteristics of an aggregated outbreak of acute hemorrhagic conjunctivitis in a school in Bao'an District,Shenzhen in 2023,and to analyze its virological characteristics,providing a reference and basis for the rapid tracing of the source of infection and the handling of such outbreaks.Methods A total of 22 eye swab samples were collected from an AHC cluster in a school in Bao'an District,Shenzhen in 2023.Real-time fluorescence quantitative polymerase chain reaction(PCR)method was used to detect Enterovirus 70(EV70)and Coxsackievirus A24(CVA24)viruses in eye swab specimens collected from patients.The positive samples were performed by full sequencing of the viral gene(VP1 nucleotide).The sequenced data was compared with that from different periods and regions for CVA24,analyzing homologies and constructing a phylogenetic tree.Additionally,the nucleotide sequences were translated into amino acid sequences to analyze mutations at the amino acid sites.Results Among the 22 eye swab specimens collected,16 samples were positive for CVA24 virus as determined by fluorescence quantitative PCR,with a positive rate of 72.73%,while all samples tested negative for EV70.VP1 primers were used to amplify 16 positive specimens,successfully sequencing the complete VP1 sequence from 14 samples.They were determined to be of the CVA24v GⅣgenotype.The genetic evolutionary analysis showed that the 14 CVA24v nucleotide sequences had 100.00%homology with each other,85.14%homology with the prototype strain EH24/70 of CVA24v,and 77.27%homology with the prototype strain Joseph of CVA24.The phylogenetic tree showed that these isolates and the CVA24v strains circulating in Zhongshan City,Guangdong Province at the same time were on the same evolutionary clade,suggesting a close phylogenetic relationship.Compared with the amino acid sequence of the reference sequence before 2023,the amino acid sequence of the VP1 region of this epidemic strain formed a new mutation at the 16th site,with a substitution from leucine(L)to isoleucine(I).Conclusions The pathogen causing this outbreak of acute hemorrhagic conjunctivitis was the Coxsackievirus A24 variant,presenting a new variant site appears in its amino acid sequence.According to the results of the phylogenetic tree,the strain is presumed to originate from a strain circulating in Guangdong Province during the same period.